Method for enhanced uptake of viral vectors in the myocardium

ABSTRACT

The present invention relates to improved therapies for the treatment of heart disease, particularly the improved delivery of therapeutic agents to heart tissue by direct infusion into the coronary circulation. A preferred embodiment of the invention is a method of treating or preventing a cardiovascular disease by transfecting cardiac cells of a large mammal, the method comprising, identifying a mammal in need of treatment or prevention of heart disease, supplying NO to the coronary circulation prior to, and/or during the infusion of a therapeutic polynucleotide into a blood vessel of the coronary circulation in vivo, where the therapeutic polynucleotide is infused into the blood vessel over a period of at least about three minutes, where the coronary circulation is not isolated or substantially isolated from the systemic circulation of the mammal; and where the therapeutic polynucleotide transfects cardiac cells of the animal resulting in the treatment or prevention of the heart disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a non-provisional of U.S. Provisional PatentApplication Ser. No. 61/029,881, filed Feb. 19, 2008, from whichpriority is claimed under 35 USC section 119(e)(1), and whichapplication is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to gene therapies for the treatment ofheart diseases, particularly the enhanced delivery of polynucleotides toheart tissue.

2. Description of the Related Art

Heart disease is a major public health issue of very high prevalence,especially in the Western world. Cardiac conditions include coronaryartery disease, ischemic heart disease, angina, heart failure, valvularheart disease, cardiac arrhythmias and cardiac inflammation(myocarditis) to name a few. Coronary artery disease and heart failureare possibly the most serious and prevalent, together being a leadingcause of death in the Western world. The impact of acute myocardialinfarction and congestive heart failure and their sequelae on thequality of life of patients and the cost of health care drives thesearch for new therapies.

Heart failure (HF) is a serious condition in which the heart loses itsability to pump blood efficiently. Data from the National Heart, Lungand Blood Institute, suggests about 5 million people in the UnitedStates alone have heart failure, and another 550,000 new cases arediagnosed each year. HF contributes to or causes about 300,000 deathsannually. The disease is most common in people aged 65 or older, womenand African Americans. The most common symptoms of heart failure areshortness of breath, feeling tired, and swelling in the ankles, feet,legs, and sometimes the abdomen. There is no cure for congestive heartfailure, and a clear need exists in the art for effective therapies.

One method of treating heart disease, such as HF, which has begun toreceive more attention is gene therapy, wherein a polynucleotide isdelivered to the cardiac tissue, typically in a viral vector. Numerousmeans of delivering viral vector to the heart have been attempted,including direct injection into the heart muscle (Liu et al., FASEB J.2006; 20(2):207-16; Li et al. Toxicol. Appl. Pharmacol. Jan. 25, 2006(electronic publication); Zhu et al., Circulation. 2005;112(17):2650-9), intracoronary delivery (Nykanen et al., Circ. Res.2006; 98(11):1373-80; Kaspar et al., J. Gene Med. 2005; 7(3):316-24),catheter-based antegrade intracoronary delivery with coronary venousblockade (Hayase et al., Am. J. Physiol. Heart Circ. Physiol. 2005;288(6):H2995-3000), aortic and pulmonary artery cross clamping followedby proximal aortic injection of adeno-associated viral vector (Kaspar etal., J. Gene Med. 2005; 7(3):316-24). Leiden and Svensson mention invivo infusion of a rAAV vector into a coronary artery or sinusgenerally, but only describe in detail the perfusion of a mouse heartwith a reporter gene ex vivo at 4° C. where the heart has stoppedbeating (WO 00/38518)—a method that is impractical for the treatment oflarge mammals, such as humans.

Thus, these methods are all inadequate for use in a clinical setting,for example because these methods are too risky due to the need forsurgical intervention or interruption of flow of oxygenated blood to theheart muscle, because of the amount of viral vector required to practicethe method, because of the low percentage of tissue transfected, becausethe fact that transduction is limited to the site of theinjection/administration only, or because the method is not practical orunproven for the treatment of disease in large animals or humans. Thereremains a need for a simple, minimally invasive, yet effective means ofdelivering transgenes using viral vectors to cardiac tissue to treat adisease, particularly in humans.

For instance, previously described in U.S. patent application Ser. No.11/778,900 (incorporated herein by reference in its entirety) is amethod of transfection of cardiac cells using a slow infusion of atherapeutic polynucleotide into coronary vessels. Increasing theefficiency of transfection of cardiac cells with the polynucleotide canlead to an increase in the efficacy of the treatment.

Use of nitroglycerin as part of a pretreatment cocktail has been used inmyocardial gene transfer therapy in animals (see Sasano T., et al.,“Targeted High-Efficiency, Homogenous Myocardial Gene Transfer” in J.Mol. Cell. Cardiol., May 2007; 42(5):954-961, which is herebyincorporated by reference in its entirety, describes pig experimentsinvolving myocardial gene transfer using a viral vector). In an effortto increase the efficiency of gene transfer in normal healthy pigs, theyreport using a pretreatment cocktail comprising vascular endothelialgrowth factor (VEGF), nitroglycerin, adenosine and calcium, followed bydosing with the viral vector and combinations of the above mentionedagents. However, their treatment protocols are not clinically practicalas such protocols would lead to prohibitive hypotension and cardiacside-effects. For example, Sasano reports that “infusion of thepretreatment and virus solutions caused an immediate systolic bloodpressure decrease of 30 mmHg that stabilized within the first minute ofperfusion. The average heart rate also decreased to 50-60/min thenstabilized over the same time course.” The authors reported further,“ventricular fibrillation (VF) occurred during coronary infusion in 5 ofthe first 10 pigs (50%) and 4 out of remaining 71 pigs (5.6%)” (Sasanoet al. at pg. 958). Given the frail status of most human subjects withadvanced cardiac disease, these side effects are likely even lesstolerable than in young, normal, healthy animals.

Thus, there still exists a need to develop a treatment method forincreasing the efficiency of cardiac transfection using vasodilation andviral vectors such as adeno-associated virus (AAV) that can be used in aclinical setting, without incurring life-threatening hypotension orcardiac arrhythmias.

SUMMARY OF THE INVENTION

The present invention relates to uses and therapies for the treatment ofheart diseases, particularly to improving or enhancing the delivery oftherapeutic agents to heart tissue by the use of a vasodilator agent,preferably a nitric oxide (NO) increasing substance, by directintracoronary, intravenous, or subcutaneous injection or infusion, ororal administration, without the need for obstructing blood flow.

A preferred embodiment of the invention is a method of treating orpreventing a cardiovascular disease by transfecting cardiac cells of alarge mammal, the method comprising identifying a mammal in need oftreatment or prevention of a cardiovascular disease; administering avasodilating substance to said mammal sufficient to dilate a bloodvessel of the coronary circulation; and administering a therapeuticpolynucleotide into a blood vessel of the coronary circulation in vivo;wherein said therapeutic polynucleotide is infused into said bloodvessel over a period of at least about three minutes, wherein thecoronary circulation is not isolated or substantially isolated from thesystemic circulation of the mammal, and wherein said therapeuticpolynucleotide transfects cardiac cells of said mammal resulting in thetreatment or prevention of said cardiovascular disease. In someembodiments, said vasodilating substance is a NO increasing substance.In some embodiments, said NO increasing substance is nitroglycerin.

In some embodiments, said NO increasing substance is administered into ablood vessel of the coronary circulation. In some embodiments, said NOincreasing substance is administered in a manner selected from the groupconsisting of: prior to said infusion of said therapeuticpolynucleotide, concurrently with said infusion of said therapeuticpolynucleotide, and prior to and concurrently with said infusion of saidtherapeutic polynucleotide. In some embodiments, said NO increasingsubstance is administered as a bolus injection not more than 5 minutesprior to said infusion of said therapeutic polynucleotide.

In some embodiments, said NO increasing substance is administered as abolus injection not more than 5 minutes prior to said infusion of saidtherapeutic polynucleotide and said NO increasing substance is infusedinto said blood vessel concurrently with said infusion of saidtherapeutic polynucleotide over a period of at least about 10 minutes.

In some embodiments, the NO increasing substance is about 50 μg to about150 μg of nitroglycerin.

In some embodiments, administration of said NO increasing substancecomprises antegrade epicardial coronary artery injection of 1.5 mL of a100 μg/mL solution of nitroglycerin into at least one of a left or rightcoronary artery via percutaneous catheter over a period of less than 1minute, wherein said administration of said NO increasing substance isless than 3 minutes prior to said infusion of said therapeuticpolynucleotide, and wherein no other vasodilator or vascular permeationenhancer is administered to said mammal. In some embodiments, the methodfurther comprises infusing nitroglycerin into said blood vesselconcurrently with said infusion of said therapeutic polynucleotide. Insome embodiments, said mammal is a human and said cardiovascular diseaseis heart failure, wherein said therapeutic polynucleotide is packaged ina DNAse resistant particle (DRP) of a AAV2/1 viral vector, and a totalnumber of DRP infused into said blood vessel is not more than about1×10¹³, wherein the therapeutic polynucleotide comprises a SERCA2acoding sequence, wherein said blood vessel is at least one of the leftor right coronary artery, and wherein said infusion of said therapeuticpolynucleotide lasts at least about 10 minutes. In some embodiments,said treatment improves a measurement of absolute ejection fraction ofsaid human's heart six months after said treatment as compared to ameasurement of absolute ejection fraction of said human's heart prior tosaid treatment.

In some embodiments, said NO increasing substance is administeredsystemically. In some embodiments, said NO increasing substance isadministered systemically in a manner selected from the group consistingof: intravenous injection, intravenous infusion, oral administration,transdermal administration, and subcutaneous administration.

In some embodiments, said NO increasing substance is administered in amanner selected from the group consisting of: prior to said infusion ofsaid therapeutic polynucleotide, concurrently with said infusion of saidtherapeutic polynucleotide, and prior to and concurrently with saidinfusion of said therapeutic polynucleotide.

In some embodiments, about 0.5 mg to about 2.5 mg of nitroglycerin isadministered by intravenous infusion over a period of at least 30minutes prior to said infusion of said therapeutic polynucleotide,wherein said infusion of said therapeutic polynucleotide begins withinnot more than three minutes of the completion of said intravenousinfusion of nitroglycerin, and wherein no other vasodilator or vascularpermeation enhancer is administered to said mammal. In some embodiments,the method further comprises infusing an additional amount ofnitroglycerin concurrently with said infusion of said therapeuticpolynucleotide. In some embodiments, said mammal is a human and saidcardiovascular disease is heart failure, wherein said therapeuticpolynucleotide is packaged in a DNAse resistant particle (DRP) of aAAV2/1 viral vector, and a total number of DRP infused into said bloodvessel is not more than about 1×10¹³, wherein the therapeuticpolynucleotide comprises a SERCA2a coding sequence, wherein said bloodvessel is at least one of the left or right coronary artery, and whereinsaid infusion of said therapeutic polynucleotide lasts at least about 10minutes. In some embodiments, said treatment improves a measurement ofabsolute ejection fraction of said human's heart six months after saidtreatment as compared to a measurement of absolute ejection fraction ofsaid human's heart prior to said treatment.

An embodiment of the invention is a therapeutic polynucleotide for usein a method of treating or preventing a cardiovascular disease bytransfecting cardiac cells of a large mammal, wherein the methodcomprises dilating a blood vessel of the coronary circulation byadministering a vasodilating substance to said mammal prior to, and/orconcurrent with, administering said therapeutic polynucleotide. In someembodiments, the method comprises administering the therapeuticpolynucleotide into a blood vessel of the coronary circulation in vivo,wherein said therapeutic polynucleotide is infused into said bloodvessel over a period of at least about three minutes, wherein thecoronary circulation is not isolated or substantially isolated from thesystemic circulation of the mammal, and wherein said therapeuticpolynucleotide transfects cardiac cells of said mammal resulting in thetreatment or prevention of said cardiovascular disease.

In some embodiments, said vasodilating substance is a NO increasingsubstance. In some embodiments, said NO increasing substance isadministered in a manner selected from the group consisting of: prior tosaid infusion of said therapeutic polynucleotide, concurrently with saidinfusion of said therapeutic polynucleotide, and prior to andconcurrently with said infusion of said therapeutic polynucleotide.

In some embodiments, said NO increasing substance is administered into ablood vessel of the coronary circulation. In some embodiments, said NOincreasing substance is administered as a bolus injection not more than5 minutes prior to said infusion of said therapeutic polynucleotide andwherein said NO increasing substance is infused into said blood vesselconcurrently with said infusion of said therapeutic polynucleotide overa period of at least about 10 minutes.

In some embodiments, the NO increasing substance is about 50 μg to about150 μg of nitroglycerin.

In some embodiments, said administration of said NO increasing substancecomprises antegrade epicardial coronary artery injection of 1.5 mL of a100 μg/mL solution of nitroglycerin into at least one of a left or rightcoronary artery via percutaneous catheter over a period of less than 1minute, wherein said administration of said NO increasing substance isless than 3 minutes prior to said infusion of said therapeuticpolynucleotide, and wherein no other vasodilator or vascular permeationenhancer is administered to said mammal. In some embodiments, the methodfurther comprises infusing nitroglycerin into said blood vesselconcurrently with said infusion of said therapeutic polynucleotide. Insome embodiments, said mammal is a human and said cardiovascular diseaseis heart failure, wherein said therapeutic polynucleotide is packaged ina DNAse resistant particle (DRP) of a AAV2/1 viral vector, and a totalnumber of DRP infused into said blood vessel is not more than about1×10¹³, wherein the therapeutic polynucleotide comprises a SERCA2acoding sequence, wherein said blood vessel is at least one of the leftor right coronary artery, and wherein said infusion of said therapeuticpolynucleotide lasts at least about 10 minutes. In some embodiments,said method of treating or preventing improves a measurement of absoluteejection fraction of said human's heart six months after said treatmentas compared to a measurement of absolute ejection fraction of saidhuman's heart prior to said treatment.

In some embodiments, said NO increasing substance is administeredsystemically in a manner selected from the group consisting of:intravenous injection, intravenous infusion, oral administration,transdermal administration, and subcutaneous administration. In someembodiments, said administration of said NO increasing substancecomprises administering about 0.5 mg to about 2.5 mg of nitroglycerin byintravenous infusion over a period of at least 30 minutes prior to saidinfusion of said therapeutic polynucleotide, wherein said infusion ofsaid therapeutic polynucleotide begins within not more than threeminutes of the completion of said intravenous infusion of nitroglycerin,and wherein no other vasodilator or vascular permeation enhancer isadministered to said mammal. In some embodiments, said method furthercomprises infusing an additional amount of nitroglycerin concurrentlywith said infusion of said therapeutic polynucleotide. In someembodiments, said mammal is a human and said cardiovascular disease isheart failure, wherein said therapeutic polynucleotide is packaged in aDNAse resistant particle (DRP) of a AAV2/1 viral vector, and a totalnumber of DRP infused into said blood vessel is not more than about1×10¹³, wherein the therapeutic polynucleotide comprises a SERCA2acoding sequence, wherein said blood vessel is at least one of the leftor right coronary artery, and wherein said infusion of said therapeuticpolynucleotide lasts at least about 10 minutes. In some embodiments,said method of treating or preventing improves a measurement of absoluteejection fraction of said human's heart six months after said treatmentas compared to a measurement of absolute ejection fraction of saidhuman's heart prior to said treatment.

Another embodiment of the invention is the use of a therapeuticpolynucleotide for the manufacture of a medicament for treating orpreventing a cardiovascular disease in a large mammal, wherein thetherapeutic polynucleotide transfects cardiac cells of said large mammalresulting in the treatment or prevention of said cardiovascular disease,and wherein said medicament is for administration in combination with avasodilating substance that dilates a blood vessel of the coronarycirculation of said mammal prior to, and/or concurrent with,administration of said medicament. In some embodiments, theadministration of the medicament comprises administering the therapeuticpolynucleotide into a blood vessel of the coronary circulation in vivo,wherein said therapeutic polynucleotide is infused into said bloodvessel over a period of at least about three minutes, and wherein thecoronary circulation is not isolated or substantially isolated from thesystemic circulation of the mammal. In some embodiments, saidvasodilating substance is a nitric oxide (NO) increasing substance. Insome embodiments, said NO increasing substance is administered in amanner selected from the group consisting of: prior to said infusion ofsaid therapeutic polynucleotide, concurrently with said infusion of saidtherapeutic polynucleotide, and prior to and concurrently with saidinfusion of said therapeutic polynucleotide.

In some embodiments, said NO increasing substance is administered into ablood vessel of the coronary circulation. In some embodiments, said NOincreasing substance is administered as a bolus injection not more than5 minutes prior to said infusion of said therapeutic polynucleotide andwherein said NO increasing substance is infused into said blood vesselconcurrently with said infusion of said therapeutic polynucleotide overa period of at least about 10 minutes. In some embodiments, the NOincreasing substance is about 50 μg to about 150 μg of nitroglycerin.

In some embodiments, said administration of said NO increasing substancecomprises antegrade epicardial coronary artery injection of 1.5 mL of a100 μg/mL solution of nitroglycerin into at least one of a left or rightcoronary artery via percutaneous catheter over a period of less than 1minute, wherein said administration of said NO increasing substance isless than 3 minutes prior to said infusion of said therapeuticpolynucleotide, and wherein no other vasodilator or vascular permeationenhancer is administered to said mammal. In some embodiments, the methodfurther comprises infusing nitroglycerin into said blood vesselconcurrently with said infusion of said therapeutic polynucleotide. Insome embodiments, said mammal is a human and said cardiovascular diseaseis heart failure, wherein said therapeutic polynucleotide is packaged ina DNAse resistant particle (DRP) of a AAV2/1 viral vector, and a totalnumber of DRP infused into said blood vessel is not more than about1×10¹³, wherein the therapeutic polynucleotide comprises a SERCA2acoding sequence, wherein said blood vessel is at least one of the leftor right coronary artery, and wherein said infusion of said therapeuticpolynucleotide lasts at least about 10 minutes. In some embodiments,said method of treating or preventing improves a measurement of absoluteejection fraction of said human's heart six months after said treatmentas compared to a measurement of absolute ejection fraction of saidhuman's heart prior to said treatment.

In some embodiments, said NO increasing substance is administeredsystemically in a manner selected from the group consisting of:intravenous injection, intravenous infusion, oral administration,transdermal administration, and subcutaneous administration. In someembodiments, administration of said NO increasing substance comprisesadministering about 0.5 mg to about 2.5 mg of nitroglycerin byintravenous infusion over a period of at least 30 minutes prior to saidinfusion of said therapeutic polynucleotide, wherein said infusion ofsaid therapeutic polynucleotide begins within not more than threeminutes of the completion of said intravenous infusion of nitroglycerin,and wherein no other vasodilator or vascular permeation enhancer isadministered to said mammal. Some embodiments further comprise infusingan additional amount of nitroglycerin concurrently with said infusion ofsaid therapeutic polynucleotide. In some embodiments, said mammal is ahuman and said cardiovascular disease is heart failure, wherein saidtherapeutic polynucleotide is packaged in a DNAse resistant particle(DRP) of a AAV2/1 viral vector, and a total number of DRP infused intosaid blood vessel is not more than about 1×10¹³, wherein the therapeuticpolynucleotide comprises a SERCA2a coding sequence, wherein said bloodvessel is at least one of the left or right coronary artery, and whereinsaid infusion of said therapeutic polynucleotide lasts at least about 10minutes. In some embodiments, said method of treating or preventingimproves a measurement of absolute ejection fraction of said human'sheart six months after said treatment as compared to a measurement ofabsolute ejection fraction of said human's heart prior to saidtreatment.

In some of the embodiments, said NO increasing substance comprisesnitroglycerin. In some embodiments, said NO increasing substanceconsists essentially of nitroglycerin. In some embodiments, said NOincreasing substance consists of nitroglycerin. In some embodiments, noother vasodilator or vascular permeation enhancer is administered tosaid mammal.

In some embodiments, the outflow of the coronary circulation is notnonnaturally restricted.

In some embodiments, transfection of cardiac cells of the anteriorlateral ventricle, inferior lateral ventricle, septum and rightventricle is detectable using quantitative PCR (RNA or DNA).

In some embodiments, the polynucleotide is capable of expressing aprotein capable of modulating a cellular activity of the cardiac cells.In some embodiments, said cellular activity is a calcium cycling pathwayof a cardiomyocyte. In some embodiments, said protein is asarcoplasmic/endoplasmic reticulum ATPase (SERCA). In someembodiments,the SERCA is SERCA2a.

In some embodiments, said polynucleotide is present in a viral vectorselected from the group consisting of an adeno-associated virus, anadenovirus, a retrovirus, a herpes simplex virus, a bovine papillomavirus, a lentiviral vector, a vaccinia virus, and a polyoma virus. Insome embodiments, said viral vector is AAV virus. In some embodiments,said viral vector is AAV virus comprising heterologous capsid proteinssuch that capsid proteins VP1, VP2 and VP3 are not all of the sameserotype AAV. In some embodiments, said heterologous capsid proteinscomprise capsid proteins from AAV1 and AAV2. In some embodiments, saidviral vector is an AAV2/1 vector. In some embodiments, saidpolynucleotide is operably linked to a CMV-based promoter and packagedin said viral vector. In some embodiments, said polynucleotide comprisesa SERCA2a coding sequence.

In some embodiments, said transfection of said cardiac cells increaseslateral ventricle fractional shortening. In some embodiments, saidmammal is human and said disease is congestive heart failure. In someembodiments, said transfection of said cardiac cells increases lateralventricle fractional shortening when measured about 4 months after saidinfusion by at least 25% as compared to lateral ventricle fractionalshortening before infusion of the polynucleotide. In some embodiments,said transfection of said cardiac cells results in an improvement in ameasure of cardiac function selected from the group consisting ofexpression of SERCA2a protein, fractional shortening, ejection fraction,cardiac output, time constant of ventricular relaxation, and regurgitantvolume.

In some embodiments the infusion into the blood vessel is at a rate ofless than or equal to about 6.0 mL/min, in some it is at a rate of lessthan or equal to about 2.5 mL/min, in some it is at a rate of less thanor equal to about 2.0 mL/min, in some it is at a rate of less than orequal to about 1.2 mL/min, in some it is at a rate of less than or equalto about 1.0 mL/min, in some is at a rate of less than or equal to about0.6 mL/min.

In a preferred embodiment, the polynucleotide is present in a viralvector selected from the group consisting of an adeno-associated virus,an adenovirus, a retrovirus, a herpes simplex virus, a bovine papillomavirus, a lentiviral vector, a vaccinia virus, and a polyoma virus. In amore preferred embodiment, the viral vector is AAV virus or an AAVmolecular variant (see Li et al., Molecular Therapy vol. 16 no. 7 Jul.2008, pg. 1252-1260, incorporated herein by reference in its entirety),and in a more preferred embodiment the viral vector is an AAV2/1 vector.The AAV2/1 vector consists of an AAV serotype 1 capsid and InvertedTerminal Repeats (ITRs) derived from AAV serotype 2. In someembodiments, the polynucleotide is operably linked to a CMV-basedpromoter and packaged in the viral vector. In a preferred embodiment,the polynucleotide comprises a human SERCA2a cDNA. Preferably, thevector consists of an AAV serotype 1 capsid and contains the humanSERCA2a cDNA flanked by Inverted Terminal Repeats (ITRs) derived fromAAV serotype 2 (AAV1 /SERCA2a).

A preferred embodiment of the invention is a method of treating orpreventing a heart disease by transfecting cardiac cells of a largemammal, the method comprising: injecting between 50 and 150 microgramsof nitroglycerin via intracoronary bolus injection lasting less than oneminute into a coronary blood vessel; infusing between about 1.4×10¹¹ toabout 1×10¹³ DRP of AAV1/SERCA2a into a blood vessel of the coronarycirculation in vivo, where the AAV1/SERCA2a is infused into the bloodvessel over a period of at least about three minutes, where the coronarycirculation is not isolated or substantially isolated from the systemiccirculation of the mammal; and where the AAV1/SERCA2a transfects cardiaccells of the mammal resulting in the treatment or prevention of theheart disease. In a preferred embodiment, nitroglycerin is the onlyvasodilator or permeability enhancer administered, and no othervasodilator or permeability enhancer is administered.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Map of heart sections tested for expression of SERCA2a in normalGöttingen Minipigs. (A) basal layer of left ventricle (LV), (B) middlelayer of LV, (C) apical layer of LV.

FIG. 2. Results from a 30 day, single dose, cardiac distribution studyof direct intracoronary infusion of AAV1/SERCA2a in normal GöttingenMinipigs. Graphs demonstrate SERCA2a mRNA expression via RT-PCR in heartsections at a 1-month of follow-up from Groups 1 and 3.

FIG. 3. Results from a 30 day, single dose, tissue distribution study ofdirect intracoronary infusion of AAV1/SERCA2a in normal GöttingenMinipigs. Graphs demonstrate SERCA2a protein expression in heartsections at a 1-month of follow-up from Groups 1, 2, 3 and 4.

FIG. 4. The mean aortic pressures from Groups 3 and 4 duringexperimental procedure for the administration of AAV1/SERCA2a in normalGöttingen Minipigs.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present technology relates to uses and therapies for the treatmentof heart diseases, particularly to enhancing the delivery of therapeuticagents to heart tissue with vasodilators without the need forobstructing blood flow. A preferred embodiment of the invention is amethod of treating or preventing a heart disease by transfecting cardiaccells of a large mammal, the method comprising: identifying a mammal inneed of treatment or prevention of a cardiac disease; injecting orinfusing a substance to increase vasodilation preferably by increasingthe amount of nitric oxide in the coronary circulation; infusing atherapeutic polynucleotide into a blood vessel of the coronarycirculation in vivo, where the therapeutic polynucleotide is infusedinto the blood vessel over a period of at least about three minutes,where the coronary circulation is not isolated or substantially isolatedfrom the systemic circulation of the mammal; and where the therapeuticpolynucleotide transfects cardiac cells of the mammal resulting in thetreatment or prevention of the heart disease.

As used herein, “polynucleotide ” has its ordinary and customary meaningin the art and includes any polymeric nucleic acid such as DNA or RNAmolecules, as well as chemical derivatives known to those skilled in theart. Polynucleotides include not only those encoding a therapeuticprotein, but also include sequences that can be used to decrease theexpression of a targeted nucleic acid sequence using techniques known inthe art (e.g., antisense, interfering, or small interfering nucleicacids). One example is a sequence which reduces or eliminates theexpression of phospholamban. Polynucleotides can also be used toinitiate or increase the expression of a targeted nucleic acid sequenceor the production of a targeted protein within cells of thecardiovascular system. Targeted nucleic acids and proteins include, butare not limited to, nucleic acids and proteins normally found in thetargeted tissue including naturally occurring mutations, derivatives ofsuch naturally occurring nucleic acids or proteins, naturally occurringnucleic acids or proteins not normally found in the targeted tissue, orsynthetic nucleic acids or proteins. One or more polynucleotides can beused in combination, administered simultaneously and/or sequentially, toincrease and/or decrease one or more targeted nucleic acid sequences orproteins.

As used herein the terms “infusion,” “infused,” and “infusing” havetheir ordinary and customary meaning in the art and refer toadministration for a time period (typically a minute or more) that issubstantially longer than the art recognized term of “injection” or“bolus injection,” (typically less than a minute). The flow rate of theinfusion will depend at least in part on the volume administered,however the flow rate of an “infusion” is slower than that of an“injection” for the same volume.

An “effective amount” has its ordinary and customary meaning in the artand includes an amount sufficient to effect or achieve a beneficial ordesired therapeutic effect. For example, an “effective amount” is anamount that achieves any of the following: an increase in lateralventricle fractional shortening; and/or palliation, amelioration,stabilization, reversal, slowing or delay in the progression or a signor symptom of the disease state. An effective amount can be administeredin one or more administrations.

As used herein “in conjunction with,” “in combination with,”“concurrent,” or “concurrently,” have their ordinary and customarymeaning in the art and include administration of one treatment modalityin addition to another treatment modality. For example, infusion of apolynucleotide to a subject can be carried out in addition to administera pharmaceutical composition or compositions to the same individual. Asused herein, these terms include simultaneous administration, or nearlysimultaneous administration.

The disclosed methods and therapeutic agents disclosed herein can becombined with existing treatments for cardiac disease, including thoselisted above in the introduction, such as drugs and percutaneous orsurgical intervention, to provide an enhanced therapeutic effectcompared to existing treatments alone. An enhanced therapeutic effectmay be demonstrated by, for example, an extension of the time periodbetween the worsening of the signs or symptoms of the disease comparedto the average or typical time period for existing treatment regimens,or the lengthening of time required before additional treatment isrequired compared to the average or typical time for standard treatmentalone.

As used herein, “treat” or “treatment” of disease has its ordinary andcustomary meaning in the art and includes the stabilization, cure, orless than complete cure of a disease, including the halting or slowingof the progression of a disease or a sign or symptom of the disease. Theterm “prevention” has its ordinary and customary meaning in the art andincludes complete or incomplete prevention, or a delay of the onset of,a disease or a sign or symptom of a disease. The terms “therapeutic,”“therapeutic effect” or “clinical effect” includes both treatment andprevention. Examples of diseases intended to be treated using thepresent technology that are associated with the cardiovascular systeminclude, but are not limited to, heart failure, ischemia, arrhythmia,myocardial infarction, congestive heart failure, transplant rejection,abnormal heart contractility, non-ischemic cardiomyopathy, mitral valveregurgitation, aortic stenosis or regurgitation, abnormal Ca²⁺metabolism and congenital heart disease. For example, beneficial ordesired clinical results or therapeutic effects include, but are notlimited to, increased survival, a greater alleviation of signs orsymptoms of cardiovascular disease, increased diminishment of extent ofdisease, stabilization (i.e., not worsening) of disease state, delay orslowing of disease progression, amelioration or palliation of thedisease state, and remission (whether partial or total), whetherdetectable or undetectable. Other examples of therapeutic effectinclude, but are not limited to, increased lateral ventricle fractionalshortening; augmented cardiac contractility at the cellular and intactanimal levels, reversal of cardiac remodeling, and normalization of theabnormally high diastolic levels of cytosolic calcium. Other clinicalfeatures which can be improved in a subject treated with an embodimentof the present invention include without limitation survival, cardiacmetabolism, heart contractility, heart rate, ventricular function (e.g.,left ventricular ejection fraction (LVEF), left ventricular end-systolicvolume (LVESV), end-diastolic pressure (LVEDP), left ventricularsystolic pressure (LVSP)), Ca²⁺ metabolism (e.g., intracellular Ca²⁺concentration, peak or resting [Ca²⁺], SR Ca²⁺ ATPase activity,phosphorylation state of phospholamban), force generation, relaxationand pressure of the heart, a force frequency relationship, cardiocytesurvival or apoptosis or ion channel activity (e.g., sodium calciumexchange, sodium channel activity, calcium channel activity, sodiumpotassium ATPase pump activity), activity of myosin heavy chain,troponin I, troponin C, troponin T, tropomyosin, actin, myosin lightchain kinase, myosin light chain 1, myosin light chain 2 or myosin lightchain 3, IGF-1 receptor, PI3 kinase, AKT kinase, sodium-calciumexchanger, calcium channel (L and T), calsequestrin, calreticulin,inhibitor-1 of the type 1 protein phosphatase, or any agent promotingdephosphorylation of phospholamban or inhibitor of the sarcoplasmicreticulum calcium-pump (SERCA2a). Other measures of cardiac diseasewhich can be improved include fractional shortening, cardiac output,ejection fraction, Tau, regurgitant volume, reduced hospital stays,improved quality of life, increased treadmill time, increased distanceduring 6 minute walk test, and increased maximal oxygenated consumption(VO₂max).

As used herein, “exogenous” nucleic acids or genes are those that do notoccur in nature in the vector utilized for nucleic acid transfer; e.g.,not naturally found in the viral vector, but the term is not intended toexclude nucleic acids encoding a protein or polypeptide that occursnaturally in the patient or host, e.g., SERCA.

As used herein, “cardiac cell” includes any cell of the heart that isinvolved in maintaining a structure or providing a function of the heartsuch as a cardiac muscle cell, a cell of the cardiac vasculature, or acell present in a cardiac valve. Cardiac cells include cardiomyocytes(having both normal and abnormal electrical properties), epithelialcells, endothelial cells, fibroblasts, cells of the conducting tissue,cardiac pacemaking cells, and neurons.

As used herein, “isolated,” “substantially isolated” or “largelyisolated” and their variants are terms that do not require complete orabsolute isolation of the coronary venous, cardiac, systemic venous, orsystemic circulation; rather, they are intended to mean that a majority,preferably the major part or even substantially all of the specifiedcirculation is isolated. As used herein, “partially isolated” refers toany nontrivial portion of the specified circulation being isolated.

As used herein, “nonnaturally restricted” includes any method ofrestricting the flow of fluid through a blood vessel, e.g., ballooncatheter, sutures, etc., but does not include naturally occurringrestriction, e.g. plaque build-up (stenosis). Nonnatural restrictionincludes substantial or total isolation of, for example, the coronarycirculation.

As used herein, “modulating” has its ordinary meaning, and encompassesboth increasing and decreasing the expression or activity of the target.

As used herein, the term “minimally invasive” is intended to include anyprocedure that does not require open surgical access to the heart orvessels closely associated with the heart. Such procedures include theuse of endoscopic means to access the heart, and also catheter-basedmeans relying on access via large arteries and veins, such as thefemoral artery.

As used herein, the term “adeno-associated virus” or “AAV” encompassesall subtypes, serotypes and pseudotypes, as well as naturally occurringand recombinant forms or molecular variants (see Li et al). A variety ofAAV serotypes and strains are known in the art and are publiclyavailable from sources, such as the ATCC, and academic or commercialsources. Alternatively, sequences from AAV serotypes and strains whichare published and/or available from a variety of databases may besynthesized using known techniques.

As used herein, the term “serotype” refers to an AAV which is identifiedby and distinguished from other AAVs based on capsid protein reactivitywith defined antisera. There are at least twelve known serotypes ofhuman AAV, including AAV1 through AAV12, however additional serotypescontinue to be discovered, and use of newly discovered serotypes arecontemplated. For example, AAV2 serotype is used to refer to an AAVwhich contains capsid proteins encoded from the cap gene of AAV2 and agenome containing 5′ and 3′ inverted terminal repeat (ITR) sequencesfrom the same AAV2 serotype.

A “pseudotyped” AAV refers to an AAV that contains capsid proteins fromone serotype and a viral genome including 5′ and 3′ inverted terminalrepeats (ITRs) of a different or heterologous serotype. A pseudotypedrAAV would be expected to have cell surface binding properties of thecapsid serotype and genetic properties consistent with the ITR serotype.A pseudotype rAAV may comprise AAV capsid proteins, including VP1, VP2,and VP3 capsid proteins, and ITRs from any serotype AAV, including anyprimate AAV serotype from AAV1 through AAV12, as long as the capsidprotein is of a serotype heterologous to the serotype(s) of the ITRs. Ina pseudotype rAAV, the 5′ and 3′ ITRs may be identical or heterologous.Pseudotyped rAAV are produced using standard techniques described in theart.

A “chimeric” rAAV vector encompasses an AAV vector comprisingheterologous capsid proteins; that is, a rAAV vector may be chimericwith respect to its capsid proteins VP1, VP2 and VP3, such that VP1, VP2and VP3 are not all of the same serotype AAV. A chimeric AAV as usedherein encompasses AAV wherein the capsid proteins VP1, VP2 and VP3differ in serotypes, including for example, but not limited to, capsidproteins from AAV1 and AAV2, mixtures of other parvovirus capsidproteins or comprise other virus proteins or other proteins, such as forexample, proteins that target delivery of the AAV to desired cells ortissues. A chimeric rAAV as used herein also encompasses a rAAVcomprising chimeric 5′ and 3′ ITRs. The present invention encompasseschimeric rAAV vectors that comprise ITRs from different AAV serotypes,for example AAV1 and AAV2, or a chimeric rAAV may comprise syntheticsequences.

Vasodilators

Vasodilation is the widening of blood vessels that occurs fromrelaxation of smooth muscle within the vessel walls, arteries,arterioles, veins, and venules. As a result of vasodilation, vascularresistance decreases and the flow of blood increases. Intrinsic andextrinsic factors can induce vasodilation; such factors are calledvasodilators. There are two general mechanisms that cause vasodilation:lowering of intracellular calcium and/or dephoshorylation of the myosinlight chain (MLC). These mechanisms are carried out through threegeneral pathways: hyperpolarization-mediated, cAMP-mediated, orcGMP-mediated. Thus, vasodilators may exert their effects through one ormore of these intermediary pathways. In one embodiment, a vasodilatormay include, but is not limited to, adenosine, histamine (orhistamine-inducing agents), alpha blockers, theobromine, papaverine,ethanol, tetrahydrocannabinol(THC), minoxidil, or nitric oxide(including nitric oxide increasing substances). In one embodiment, onlya single vasodilating substance is administered. In another embodiment,it is contemplated to use one or more vasodilators together,sequentially, or a combination thereof. In a preferred embodiment, thevasodilator is a nitric oxide increasing substance.

Nitric oxide (NO) is a free radical molecule, which can act as a shortlived chemical transmitter, freely diffusible across membranes. NO has avariety of physiological effects. See generally, Jeremy M. Berg, et al.(2006), Biochemistry, 6th Edition. W. H. Freeman and Company. Forexample, it is known to cause vascular dilatation by controlling smoothmuscle contractility after systemic or local delivery. In the centralnervous system, NO can affects synaptic transmission stimulatinglearning and memory capacity. As another example, NO can induce plateletaggregation in blood plasma. Because of its lipophilic nature, nitricoxide can diffuse out of its cells of origin into other nearby cells,creating a signal transduction mechanism. In the coronary arteries, NOcan activate cytosolic guanylate cyclase and stimulate cyclic guanosinemonophosphate (cGMP) formation in vascular smooth muscle cells, leadingto vasodilation.

Without limitation to any specific mechanism of action, it has beendiscovered that vasodilation of the coronary circulation, or in anartery supplying blood to the heart, can increase the efficiency oftransduction of the therapeutic agent described further below. That is,transduction efficiency of the therapeutic agent can be enhanced byadministering to the coronary circulation, cardiac artery, orsystemically, a vasodilating agent or combination of agents capable ofinducing vasodilation, preferably, with a NO increasing substance. Asused herein, “NO increasing substance” includes combinations of two,three, four, five or more compounds unless indicated otherwise, and caninclude compounds that mimic an increase in NO by activating thereceptor for NO, e.g. a NO agonist, without actually increasing theamount of NO. In some embodiments, the treatment can occur before, atleast partially during, or after treatment with the primary agent. Thus,in some embodiments, NO can be used as an adjuvant to increase theefficiency, efficacy, or potency of the primary therapeutic agent.Embodiments include combinations of two, three, four, five, or more NOincreasing substances.

Increasing levels of NO, even temporarily, in the coronary circulationcan be accomplished by a variety of known techniques. As used herein, NOincreasing substance includes, but is not limited to, any of thefollowing compounds or classes of compounds, or any combination of two,three, four, five or more of the following compounds or classes ofcompounds. Agents that release NO under physiological conditions havebeen in use for a long time in the management of heart diseases. Theseagents can include, by way of example only, NO donors, NO releasingmolecules, NO precursors. For example, NO donors can include nitratessuch as glyceryl trinitrate, which may also be commonly referred to as“nitroglycerin.” Other examples of nitrates include isosorbide dinitrateand isosorbide mononitrate. NO donors can also include other agents suchas those described in Megson I L, Webb D J, “Nitric oxide donor drugs:current status and future trends” in Expert. Opin. Investig. Drugs, May2002;11(5):587-601, which is hereby incorporated by reference in itsentirety. NO releasing molecules can also increase the levels of NO inthe coronary circulation or a coronary artery. For example, NO releasingmolecules can include diazeniumdiolates or NO releasing non-steroidalanti-inflammatory drugs (NO-NSAID). NO precursors, such as L-arginine,can also be used to increase levels of NO. Other nitric oxidideincreasing substances that can be used include molecular nitric oxide,nicorandil, and nitric oxide synthase, sodium nitroprusside, andpentaerythritol tetranitrate (PETN). Moreover, agents that increase theeffects of NO are also contemplated such as phosphodiesterase type 5(PDE5) inhibitors including, but not limited to, sildenafil, tadalafil,and vardenafil.

In some embodiments, the substance used to increase the amount of nitricoxide in the coronary circulation comprises a nitric oxide donor. Insome embodiments, the nitric oxide donor comprises a nitrate. In apreferred embodiment, the nitrate comprises glyceryl trinitrate. In someembodiments, the nitrate comprises an agent selected from the groupconsisting of pentaerythritol tetranitrate, isosorbide dinitrate andisosorbide-mononitrate. In some embodiments, the nitric oxide donorcomprises sodium nitroprusside. In some embodiments, the substance usedto increase the amount of nitric oxide in the coronary circulationcomprises a nitric oxide releasing molecule. In some embodiments, thenitric oxide releasing molecule comprises an agent selected from thegroup consisting of a diazeniumdiolates and a nitric oxide-releasingnon-steroidal anti-inflammatory drugs. In some embodiments, thesubstance used to increase the amount of nitric oxide in the coronoarycirculation comprises an agent selected from the group consisting ofmolecular nitric oxide, nicorandil, and nitric oxide synthase. In someembodiments, the substance used to increase the amount of nitric oxidein the coronary circulation comprises a nitric oxide precursor. In someembodiments, the nitric oxide precursor comprises L-arginine.

Vasodilating Substance Administration

The vasodilating substance or substances can be administeredsystemically, for example orally, including but not limited tosublingual and translingual administration, transdermally, including butnot limited to via a patch or ointment, or by intravenous injection orinfusion. In a preferred embodiment, the vasodilating substance orsubstances can be administered by intracoronary injection or infusion.In another preferred embodiment, the vasodilating substance orsubstances can be administered by intravenous infusion or injection. Thefollowing sections describe further these modes of delivery.

The coronary circulation provides blood supply to the tissue of theheart. Intracoronary administration is accomplished by injection orinfusion into one or more blood vessel of the coronary circulation ofthe beating heart in vivo. There are a number of coronary arteries.Normally, four main coronary arteries provide oxygenated blood to theheart for distribution throughout the heart tissue: the left main andright coronary arteries, the left anterior descending artery, and theleft circumflex artery. Injection or infusion of one or a combination ofthese arteries is contemplated, for example injection or infusion intothe left and right coronary arteries. In one embodiment, ⅔ of the totalamount of a vasodilating substance or substances, including but notlimited to a NO increasing substance or substances, is delivered to oneblood vessel of the heart, and ⅓ is administered to another blood vesselof the heart. In another embodiment, more than 2 coronary blood vesselsare injected or infused, (e.g. 3, 4, 5 or more), and the portion oftotal volume or amount of vasodilator administered per blood vessel canbe adjusted as appropriate. The preferred embodiment utilizes antegrade,epicardial injection, or infusion, of the left and right main coronaryarteries. Also contemplated is retrograde injection or infusion of acoronary artery, or a combination of one or more antegrade andretrograde coronary arteries or veins.

Injection or infusion of one or more vasodilating substances, includingbut not limited to a NO increasing substance(s), into the coronary bloodvessel(s) is performed using standard guide-wires, catheters andinfusion pumps as needed. In a preferred embodiment, the injection orinfusion catheter is directed to the coronary artery under fluoroscopicguidance via the femoral artery. As used herein, “blood vessel of thecoronary circulation,” “coronary blood vessel” or “blood vessel of theheart” includes grafts onto coronary blood vessels, for example thoseresulting from bypass surgery. As used herein, “epicardial” refers toblood vessels located on the outer portion of the heart, e.g. the leftor right coronary arteries.

The amount of the vasodilating substance administered to the subjectwill depend on the size of the subject and the route of administration.In a preferred embodiment, the vasodilating substance or substances,including but not limited to a NO increasing substance or substances, isinjected as a single bolus injection (typically in a volume of 0.1-2 mL,in less than a minute) directly into a coronary artery less than about 5minutes prior to the administration of the viral vector or othertherapeutic agent. In some embodiments, the vasodilator(s), including NOincreasing substance(s), is administered locally or systemically,preferably by injection or infusion, at a time prior to theadministration of the viral vector or therapeutic agent that is, isabout, is at least, is at least about, is not more than, or is not morethan about, 0.5, 1, 2, 3, 4, 5, 7, 10, 12, 15, 20, 25, or 30 minutes, 1,2, 3 or more hours, or a range defined by any two of the precedingvalues. In a preferred embodiment, the range is 0.5-10 minutes. Morepreferably, the vasodilator(s) or NO increasing substance(s) isadministered, preferably by a single bolus injection, immediately priorto the administration of the viral vector or therapeutic agent. In someembodiments, where the vasodilator(s) is administered by infusion,administration of the viral vector or therapeutic agent begins at a timefollowing the end of the infusion of the vasodilator that is, is about,is at least, is at least about, is not more than, or is not more thanabout, 0.5, 1, 2, 3, 4, 5, 7, 10, 12, 15, 20, 25, or 30 minutes, 1, 2, 3or more hours, or a range defined by any two of the preceding values.

In some embodiments, the vasodilator(s) or NO increasing substance(s) isinjected or infused prior to the viral vector or therapeutic agent asdescribed herein, and a second dose of the same or differentvasodilator(s) or NO increasing substance(s) is administeredconcurrently with the viral vector or therapeutic agent, preferably overa period of at least 3 minutes, more preferably about 4 to about 10minutes, or as described in more detail below. In other embodiments, nopretreatment with the vasodilator(s) or NO increasing substance(s) isgiven, and the vasodilator(s) is administered concurrently with theviral vector or therapeutic agent which is administered as describedherein. In some embodiments, the concurrent administration isadministration of the vasodilator in the same solution as thetherapeutic agent. In other embodiments, the concurrent administrationis via different routes of administration for the vasodilator and thetherapeutic agent (e.g. intravenous and intracoronary, respectively).

In some embodiments, one or more vasodilator(s), including but notlimited to NO increasing substance(s), are administered after the viralvector or therapeutic agent. This post-administration can be in additionto pretreatment and/or concurrent administration with the therapeuticsubstance, and can be the same or different vasodilator(s) asadministered in the pretreatment and/or concurrent administration. Insome embodiments, the vasodilator(s) or NO increasing substance(s) isadministered, preferably by injection or infusion, at a time after theadministration of the viral vector or therapeutic agent that is, isabout, is at least, is at least about, is not more than, or is not morethan about 0.5, 1, 2, 3, 4, 5, 7, 10, 12, 15, 20, 25, or 30 minutes, 1,2, 3 or more hours, or a range defined by any two of the precedingvalues. In a preferred embodiment, the range is 0.5-10 minutes afteradministration of the viral vector.

In a preferred embodiment, the NO increasing substance is nitroglycerin,and the total amount of nitroglycerin administered via intracoronaryinjection or infusion, in one or more doses as described herein, is fromabout 50 μg to about 500 μg, more preferably from about 100 μg to about150 μg. The contemplated total amount, or amount per dose, ofnitroglycerin, or other vasodilator(s) or NO increasing substance(s), orcombination of substances, administered via intracoronary injection orinfusion is, is about, is at least, is at least about, is not more than,or is not more than about, 1, 2, 3, 4, 5, 7, 10, 12, 15, 20, 30, 40, 50,60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500,600, 700, 800, 900, or 1000 μg, or a range defined by any two of thepreceding values. This amount can be the amount administered as apretreatment, along with, or after the therapeutic agent, to a singlecoronary artery or all coronary arteries receiving an injection orinfusion, or any combination thereof, or can be the total amountadministered. One skilled in the art will appreciate that the dose ofvasodilating agent used for intracoronary injection or infusion isrelative to the size of the organ and not necessarily the subjects'total body weight. In one embodiment, an initial intracoronary injectionof 50 μg of nitroglycerin is given prior to infusion of the viralvector, and a second amount of 100 μg of nitroglycerin is infused withthe viral vector, preferably over at least 3 minutes, more preferablyabout 4 minutes, to about 10 minutes.

In some embodiments, the total dose of nitroglycerin administeredsystemically via intravenous injection or infusion is preferably fromabout 200 μg to about 4000 μg, more preferably from about 500 μg toabout 2500 μg. The contemplated total dose of nitroglycerin administeredvia systemic injection or infusion is, is about, is at least, is atleast about, is not more than, or is not more than about, 1, 2, 3, 4, 5,7, 10, 12, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200,250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1250, 1500,1750, 2000, 2250, 2500, 2750, 3000, 3500, or 4000 μg, or a range definedby any two of the preceding values. In some embodiments, nitroglycerinis administered intravenously, at or at about, 5 μg/minute, increased by5 μg/minute every 3-5 minutes to 20 μg/minute; if there is no responseat 20 μg/minute, the dose may be increased by 10 μg/minute every 3-5minutes up to 200 μg/minute. The contemplated dose rates ofnitroglycerin administered via systemic injection or infusion is, isabout, is at least, is at least about, is not more than, or is not morethan about, 1, 2, 3, 4, 5, 7, 10, 12, 15, 20, 30, 40, 50, 60, 70, 80,90, 100, 125, 150, 175, 200, 250, 300, 350, or 400 μg/minute or a rangedefined by any two of the preceding values. The total time of infusionof the vasodilator(s) or NO increasing substance(s) is, is about, is atleast, is at least about, is less than, is less than about, 5, 7, 10,12, 15, 20, 25, 30, 35, 40,45, 50, 55,60,65, 70, 75, 80, 85, 90,95, 100,110, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, or 600 minutes,or a range defined by any two of the preceding values. In someembodiments, IV infusion begins prior to, and continues during theadministration of the viral vector or therapeutic agent.

In some embodiments, the total dose of nitroglycerin administeredsystemically by oral means is preferably from about 5 mg to about 105mg, more preferably from about 10 mg to about 80 mg. In anotherembodiment, the preferred dose is about 15 mg to about 80 mg. Thecontemplated total dose of nitroglycerin administered orally is, isabout, is at least, is at least about, is not more than, or is not morethan about 0.4, 0.5, 0.75, 1, 2, 3, 4, 5, 7, 10, 12, 15, 20, 30, 40, 50,60, 70, 80, 90, 100, or 125 mg, or a range defined by any two of thepreceding values.

In another embodiment, the amount of nitroglycerin given systemically bysublingual administration is, is about, is at least, is at least about,is not more than, or is not more than about, 36, 54, 72, 90, 108, 126,144, 162, 180, 198, 216, 234, 252, 270, 288, 306, 324, 342, or 360 mg ora range defined by any two of the preceding values. In some embodiments,nitroglycerin is administered sublingually where the dose is, is about,is at least, is at least about, is not more than, or is not more thanabout, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65,0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or 1 mg, or a range defined by any twoof the preceding values. The sublingual dose is administered at aninterval that is, is about, is at least, is at least about, is not morethan, or is not more than about, every 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10minutes or a range defined by any two of the preceding values. In apreferred embodiment, sublingual administration of nitroglycerin isgiven from about 0.2 to about 0.6 mg every 5 minutes for a maximum of 3doses every 15 minutes. In another embodiment, nitroglycerin can begiven translingually by spray, drops, or mist. One to 2 sprays into themouth may be given every 3-5 minutes for a maximum of 3 doses in 15minutes.

In some embodiments, systemic administration of nitroglycerin can bedelivered transdermally with a transdermal patch. The total dose ofnitroglycerin administered systemically via transdermal patch ispreferably from about 2.4 mg to about 15.6 mg, more preferably fromabout 4.8 mg to about 9.6 mg. The contemplated total dose ofnitroglycerin administered via a transdermal patch is, is about, is atleast, is at least about, is not more than, or is not more than about,2.4, 3.6, 4.8, 6, 7.2, 8.4, 9.6, 10.8, 12, 13.2, 14.4, or 15.6 mg, or arange defined by any two of the preceding values. In some embodiments,nitroglycerin is administered by a transdermal patch where the dose is,is about, is at least, is at least about, is not more than, or is notmore than about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55,0.6, or 0.65 mg, or a range defined by any two of the preceding values.The transdermal patch dose is administered at an interval that is, isabout, is at least, is at least about, is not more than, or is not morethan about, every 15, 30, 45, 60, 75, or 90 minutes or a range definedby any two of the preceding values. In another embodiment, thecontemplated dose rates of nitroglycerin administered via a transdermalpatch is, is about, is at least, is at least about, is not more than, oris not more than about, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5,0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or 1 mg/hour, or arange defined by any two of the preceding values. In a preferredembodiment, nitroglycerin is given transdermally at an initial dose ofabout 0.2-0.4 mg/hour, up to doses of 0.4-0.8 mg/hour. Tolerance isminimized by using a patch-on period of 12-14 hours and a patch-offperiod of about 10-12 hours.

In another embodiment, systemic administration of nitroglycerin can bedelivered transdermally by topical application of an ointment. Thecontemplated total dose of nitroglycerin administered via topicalointment is, is about, is at least ½″ square inch upon rising and ½″square inch 6 hours later at a concentration of, or of about 0.1%,0.15%, 0.2%, 0.25%, 0.3%, 0.4%, 1%, or 2% nitroglycerin, or a rangedefined by any two of the preceding values. In preferred embodiment, theconcentration of the ointment is 0.2%. The dose may be doubled severaltimes as needed.

Some embodiments contemplate systemic delivery of nitroglycerin throughthe skin by subcutaneous injection or infusion. In some embodiments, thetotal dose of nitroglycerin administered subcutaneously is preferablyfrom about 5 mg to about 105 mg, more preferably from about 10 to about80 mg. In another embodiment, the preferred dose is about 15 mg to about80 mg. The contemplated total dose of nitroglycerin administeredsubcutaneously is, is about, is at least, is at least about, is not morethan, or is not more than about 0.4, 0.5, 0.75, 1, 2, 3, 4, 5, 7, 10,12, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 125 mg, or a rangedefined by any two of the preceding values.

In a preferred embodiment, the vasodilator(s) or NO increasingsubstance(s) or combination of substances is administered without anyother vasodilator or vascular permeation enhancing substance in anamount sufficient to increase vasodilatation or vascular permeability.In some embodiments, the NO increasing substance or combination ofsubstances, preferably nitroglycerin, is the only vasodilator orvascular permeation enhancer administered before, during and/or afterthe administration of the viral vector or therapeutic agent. In someembodiments, no vasodilator or vascular permeation enhancing substanceis administered before, during and/or after the administration of theviral vector or therapeutic agent, other than the NO increasingsubstances described herein. In an embodiment, the subject is nottreated with any vasodilator or permeation enhancing substance in anamount sufficient to enhance the uptake of a viral vector or therapeuticagent, before, during and/or after the administration of the viralvector or therapeutic agent, other than the NO increasing substancesdescribed herein, preferably nitroglycerin. Vasodilatory or permeationenhancing substances which are preferably excluded from administrationin some embodiments include, but are not limited to, vascularendothelial growth factor (VEGF), adenosine and calcium.

In some embodiments, the amount of vasodilator substance or substancesis a transfection enhancing or pharmaceutically or therapeuticallyeffective amount, wherein the amount is sufficient to enhance theefficiency of transduction of the viral vector or therapeutic agent. Theenhancement of transfection can be directly measured by examiningtransfection efficiency, or indirectly, by measuring other indicators ofsuccessful transfection such as improvements in one or more symptoms oroutcomes discussed herein. An enhancing amount is an amount thatimproves the indicator examined in comparison to the same indicator whenthe vasodilator(s) or NO increasing substance(s) is not administered.

Therapeutic Agent Administration

In a preferred embodiment of the invention, the therapeutic agent, e.g.polynucleotide/viral vector described in more detail below, isadministered to the subject by infusion into a blood vessel of thecoronary circulation of the beating heart in vivo for a period of atleast about three minutes in a particular blood vessel. In large animalmodels of the human heart and cardiovascular disease, Applicant hasfound that, unexpectedly, for administration of viral vectors arelatively long infusion time is more effective and results in superiorgene transfer efficiency into heart tissue than a bolus injection orshort (e.g., ≦1 minute) infusion time of the same amount of viralvector. The improved efficacy of infusion can be measured as a greatercopy number of the transgene per cell, increased expression of thetransgene at the mRNA and/or protein level per cell or in the tissue,and/or a greater percentage of cells of a particular tissue, e.g.cardiomyocytes, being transfected, as compared to injection. In anotherembodiment, clinical or functional measurements may be used todemonstrate the transfection efficiency from a relatively long infusiontime. Such clinical and functional assessments are described furtherherein.

Applicant has shown that this method results in successful treatment oflarge animal models of human cardiovascular disease. In addition,Applicant has discovered that by using relatively long infusion times,there is no need to isolate the coronary circulation from the systemiccirculation or otherwise re-circulate the therapeutic agent, or toartificially restrict the coronary venous circulation as a means toincrease pressure within the coronary circulation or to increase dwelltime of the therapeutic agent. Nor is there any need to cool the heart,stop the heart, or remove the heart from the animal for perfusion.Instead, Applicant's method can be practiced in a standardcatheterization lab setting using existing catheters for administration.Thus, Applicant has discovered a simple, practical, and efficaciousmeans of using gene therapy to treat cardiovascular disease in largeanimals, such as humans.

In a preferred embodiment of the invention, the therapeutic agent isadministered to the subject by infusion into a blood vessel of thecoronary circulation. The coronary circulation provides blood supply tothe tissue of the heart. There are a number of coronary arteries.Normally, four main coronary arteries provide oxygenated blood to theheart for distribution throughout the heart tissue; the left main andright coronary arteries, the left anterior descending artery, and theleft circumflex artery. Infusion of one or a combination of thesearteries is contemplated, for example infusion of the left and rightcoronary arteries. The preferred embodiment utilizes antegrade,epicardial infusion of the left and right main coronary arteries. Alsocontemplated is retrograde infusion of a coronary artery, or acombination of one or more antegrade and retrograde coronary arteries orveins. Infusion of the coronary blood vessel(s) is performed usingstandard guide-wires, catheters and infusion pumps. In a preferredembodiment, the infusion catheter is directed to the coronary arteryunder fluoroscopic guidance via the femoral artery. As used herein,“blood vessel of the coronary circulation,” “coronary blood vessel” or“blood vessel of the heart” includes grafts onto coronary blood vessels,for example those resulting from bypass surgery. As used herein,“epicardial” refers to blood vessels located on the outer portion of theheart, e.g. the left or right coronary arteries.

Once the infusion catheter is in place in the target coronary bloodvessel, the therapeutic agent is infused into the blood vessel,preferably by means of a programmable infusion pump. The amount of timetaken to infuse the therapeutic agent is an important factor inobtaining effective and superior gene transfer efficiency. Applicant hasdetermined that an infusion time of at least about 3 minutes into aparticular blood vessel is more effective than a bolus injection orshorter infusion time. Preferably, the infusion time is at least about 8minutes, more preferably at least about 10 minutes, although infusiontimes of at least about 15 minutes are contemplated. Applicant alsocontemplates that the infusion time is, is about, is at least, is atleast about, is not more than, or is not more than about, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes, orfalls within a range defined by any two of these values.

Because the infusion typically involves the use of a catheter andconnecting tubing which has a certain dead volume, the infusion deviceis often primed with a carrier solution, e.g. blood from the subject,which does not contain any therapeutic agent. Thus, the therapeuticagent is not immediately administered into the coronary circulation whenthe infusion pump is turned on. Likewise, when the syringe containingthe therapeutic agent is emptied, an amount of therapeutic agenttypically remains in the dead volume of the connecting tubing andcatheter. Immediately following the infusion of the therapeutic agent,the dead volume is flushed with an appropriate solution. The period oftime over which the therapeutic agent is actually being delivered intothe coronary circulation, as opposed to displacing dead volume in theinfusion apparatus, is the “infusion time” referred to above. Forexample, if 3 mL of therapeutic agent is loaded into an infusionapparatus with 3 mL of dead volume, and the infusion rate is 1 mL/min.,the time required to infuse the therapeutic agent into the coronarycirculation is only 3 minutes, while the total time required toadminister the 3 mL of therapeutic agent and 3 mL of dead volume is 6minutes. In some embodiments, the catheter and any connecting tubing isprimed with the therapeutic agent such that the dead volume is not anissue. Similarly, the effective amount of therapeutic agent could bedelivered without the need to flush the tubing. However, this results intherapeutic agent being left in the tubing, wasting the therapeuticagent.

Applicant contemplates that the therapeutic agent will be infused at aflow rate that is, is about, is at least, is at least about, is not morethan, or is not more than about, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2,2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0,6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 mL/min., or falls within arange defined by any two of these values. Preferably, the flow rate isbetween about 0.2 mL/min and about 6.0 mL/min., more preferably betweenabout 0.2 mL/min and about 2.5 mL/min., more preferably between about0.2 mL/min. and about 2.0 mL/min. Those of skill in the art willrecognize that delivery of the therapeutic agent is possible without aninfusion pump, however more accurate flow rates and uniform delivery arepossible with the use of an infusion pump.

The total amount of viral particles or DNase resistant particles (DRP)delivered by infusion to provide an effective amount is preferablybetween 1×10¹⁴ and about 1×10¹¹, more preferably between about 3×10¹²and 1×10¹², and more preferably about 3×10¹². However, applicant alsocontemplates that the total amount of viral particles or DRP is, isabout, is at least, is at least about, is not more than, or is not morethan about, 1×10¹⁴, 9×10¹³, 8×10¹³, 7×10¹³, 6×10¹³, 5×10¹³, 4×10¹³,3×10¹³, 2×10¹³, 1×10¹³, 9×10¹², 8×10¹², 7×10¹², 6×10¹², 5×10¹², 4×10¹²,3×10¹², 2×10¹², 1×10¹², 9×10¹¹, 8×10¹¹, 7×10¹¹, 6×10¹¹, 5×10¹¹, 4×10¹¹,3×10¹¹, 2×10¹¹, 1×10¹¹, 9×10¹⁰, 8×10¹⁰, 7×10¹⁰, 6×10¹⁰, 5×10¹⁰, 4×10¹⁰,3×10¹⁰, 2×10¹⁰, 1×10¹⁰, 9×10⁹, 8×10⁹, 7×10⁹, 6×10⁹, 5×10⁹, 4×10⁹, 3×10⁹,2×10⁹, 1×10⁹, or falls within a range defined by any two of thesevalues.

The number of DRP infused over a given time is a function of theconcentration of the solution being infused and the flow rate. The rateof DRP or viral particle infusion is preferably between about 1×10⁸/min.and about 1×10¹⁴/min., more preferably between about 5×10¹⁰/min. andabout 5×10¹²/min., more preferably between about 3×10¹⁰/min. and about1×10¹²/min., more preferably between about 6×10¹⁰/min. and about4×10¹¹/min. In a preferred embodiment, the rate of DRP or viral particleinfusion is 1×10¹¹/min., and in another preferred embodiment, it is1.25×10¹¹/min.

In one embodiment, the therapeutic agent is administered into a singleblood vessel of the heart. In another embodiment ⅔ of the total volumeof therapeutic agent is delivered to one blood vessel of the heart, and⅓ is administered to another blood vessel of the heart. In anotherembodiment, more than 2 coronary blood vessels are infused, (e.g. 3, 4,5 or more), and the portion of total infusion volume containing thetherapeutic agent administered per blood vessel can be adjusted asappropriate. The goal of the infusion is to provide diffuse, homogenousleft ventricular myocardial exposure to AAV2/1/SERCA2a via anterograde,epicardial coronary infusion. Multiple infusion scenarios exist based oncollateralization patterns, occlusive disease, and anatomic variation(e.g. post surgical bypass anatomy), but the clinician's goal is ⅓ ofAAV2/1/SERCA2a delivered to the anterolateral, ⅓ delivered to theposterolateral, and ⅓ delivered to the inferior/inferolateralmyocardium. Anatomy is defined by coronary and bypass graft angiographyto accomplish homogenous delivery to the perfused myocardium. Inaddition, one of skill in the art will recognize that while sheep andpigs are accepted animal models for human cardiovascular studies, sheepand pigs are 90% left dominant. In comparison, approximately ˜10% of thehuman population is left dominant, with the remaining 90% being right orco-dominant (Vlodaver Z. et al. Coronary Heart Disease: Clinical,Angiographic, and Pathologic Profiles. Spinger-Verlag, New York. 1976).One pathologic series suggests that 71% of patients are right dominant,17% co-dominant, 12% are left dominant (McAlpine W. Heart and CoronaryArteries. Spinger Verlag, 1975). Therefore, to achieve a similarperfusion of the left ventricle in humans vs. pigs/sheep, the optimuminfusion scenario can differ.

A ⅓ and ⅔ split of the solution volume is preferred for two bloodvessels, however the portion of the injection volume infused into aparticular blood vessel can be a volume that is, is about, is at least,is at least about, is not more than, or is not more than about, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80% of the total volume, orfalls within a range defined by any two of these values. The totalvolume of solution containing the therapeutic agent will vary accordingto the size of the animal being treated. For a human subject, a totaltherapeutic agent volume of 60 mL is preferred. However the total volumeof therapeutic agent can be a volume that is, is about, is at least, isat least about, is not more than, or is not more than about, 1, 2, 3, 4,5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 mL, orfalls within a range defined by any two of these values.

The therapeutic agents described herein can be in solution, preferably apharmaceutical composition suitable for administration directly into thecoronary circulation. The ingredients of an acceptable pharmaceuticalcomposition are known to those of skill in the art, and can include suchelements as a buffer and suitable carrier. In another embodiment, thepharmaceutical composition containing a therapeutic agent, for example aviral vector, and more preferably a AAV2/1/SERCA2a vector, is part of akit. In some embodiments, the kit contains a stock solution oftherapeutic agent and a solution for diluting the stock solution. Alsoincluded in the kit are instructions for administration of the viralvector, preferably by infusion directly into the coronary circulation asdescribed in any of the embodiments disclosed herein. The therapeuticagents and vasodilator(s), including but not limited to NO increasingsubstance(s), described herein can be used in the manufacture of amedicament for the treatment of the diseases disclosed herein, where themedicament is administered according to or in the practice of any of themethods disclosed herein.

Methods of Polynucleotide Delivery

One aspect of the present invention contemplates transfer of therapeuticpolynucleotides into a cell. Such transfer may employ viral or non-viralmethods of gene transfer. This section provides a discussion of methodsand compositions of gene or nucleic acid transfer, including transfer ofantisense, interfering, and small interfering sequences.

In one embodiment, the therapeutically significant polynucleotides areincorporated into a viral vector to mediate transfer to a cell.Additional expression constructs encoding other therapeutic agents asdescribed herein may also be transferred via viral transduction usinginfectious viral particles, for example, by transformation with anadeno-associated virus (AAV) or AAV molecular variants of the presentinvention. Alternatively, a retrovirus, bovine papilloma virus, anadenovirus vector, a lentiviral vector, a vaccinia virus, a polyomavirus, or an infective virus that has been engineered to express may beused. Similarly, nonviral methods which include, but are not limited to,direct delivery of DNA such as by perfusion, naked DNA transfection,liposome mediated transfection, encapsulation, and receptor-mediatedendocytosis may be employed. These techniques are well know to those ofskill in the art, and the particulars thereof do not lie at the crux ofthe present invention and are thus need not be exhaustively detailedherein. However, in one preferred example, a viral vector is used forthe transduction of cardiac cells to deliver a therapeuticallysignificant polynucleotide to a cell. The virus may gain access to theinterior of the cell by a specific means such receptor-mediatedendocytosis, or by non-specific means such as pinocytosis.

Adeno-Associated Virus Vectors

A preferred embodiment of the invention utilizes purified, replicationincompetent, pseudotyped recombinant adeno-associated viral (rAAV)particles. Adeno-associated viruses (AAV) are parvoviruses belonging tothe genus Dependovirus. They are small, nonenveloped, single-strandedDNA viruses which require a helper virus in order to replicate.Co-infection with a helper virus (e.g., adenovirus, herpes virus, orvaccinia virus) is necessary in order to form functionally complete AAVvirions. In vitro, in the absence of co-infection with a helper virus,AAV establishes a latent state in which the viral genome exists in anepisomal form, but infectious virions are not produced. Subsequentinfection by a helper virus “rescues” the genome, allowing it to bereplicated and packaged into viral capsids, thereby reconstituting theinfectious virion. Recent data indicate that in vivo both wild type AAVand recombinant AAV predominantly exist as large episomal concatemers.

AAV are not associated with any known human diseases, are generally notconsidered pathogenic, and do not appear to alter the physiologicalproperties of the host cell upon integration. AAV can infect a widerange of host cells, including non-dividing cells, and can infect cellsfrom different species. In contrast to some vectors, which are quicklycleared or inactivated by both cellular and humoral responses, AAVvectors have shown persistent expression in various tissues in vivo. Thepersistence of recombinant AAV vectors in non-diving cells in vivo maybe attributed to the lack of native AAV viral genes and the vector'sability to form episomal concatemers.

Adeno-associated virus (AAV) is an attractive vector system for use incell transduction because it has a high frequency of persistence as anepisomal concatemer and it can infect non-dividing cells, thus making ituseful for delivery of genes into mammalian cells, for example, intissue culture and in vivo. Studies demonstrating the use of AAV in genedelivery include Flotte et al, Proc. Natl. Acad. Sci. USA, 1993;90:10613-17 and Walsh et al., J. Clin. Invest., 1994; 94:1440-48.Recombinant AAV vectors have been used successfully for in vitro and invivo transduction of marker genes and genes involved in human diseases(see for example, Walsh et al., J. Clin. Invest. 1994; 94:1440-48). AAVhas a broad host range for infectivity. Details concerning thegeneration and use of rAAV vectors are described in U.S. Pat. No.5,139,941 and/or U.S. Pat. No. 4,797,368, each incorporated herein byreference.

Typically, recombinant AAV (rAAV) virus is made by cotransfecting aplasmid containing the gene of interest flanked by the two AAV terminalrepeats and/or an expression plasmid containing the wild-type AAV codingsequences without the terminal repeats, for example pIM45. The cells arealso infected and/or transfected with adenovirus and/or plasmidscarrying the adenovirus genes required for AAV helper function. rAAVvirus stocks made in such fashion are contaminated with adenovirus whichmust be physically separated from the rAAV particles (for example, bycesium chloride density centrifugation or column chromatography).Alternatively, adenovirus vectors containing the AAV coding regionsand/or cell lines containing the AAV coding regions and/or some or allof the adenovirus helper genes could be used. Cell lines carrying therAAV DNA as an integrated provirus can also be used.

Multiple serotypes of AAV exist in nature, with at least twelveserotypes (AAV1-AAV12) currently known. Moreover, chimeric variants havebeen produced through directed evolution (DNA shuffling) technology (seeLi et al.). Despite the high degree of homology, the different serotypeshave tropisms for different tissues. The receptor for AAV1 is unknown;however, AAV1 is known to transduce skeletal and cardiac muscle moreefficiently than AAV2. Since most of the studies have been done withpseudotyped vectors, in which the vector DNA flanked with AAV2 ITR ispackaged into capsids of alternate serotypes, it is clear that thebiological differences are related to the capsid rather than to thegenomes. Recent evidence indicates that DNA expression cassettespackaged in AAV1 capsids are at least 1 log₁₀ more efficient attransducing cardiomyocytes than those packaged in AAV2 capsids.

Engineered rAAV Vectors

In one embodiment, AAV vectors can be engineered to reduce neutralizingantibody (NAb) titers and/or cross-reactivity. Preferably, thecross-reactivity of the engineered or chimeric vector with a Nab is atleast 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 97, or 99% less than thecross-reactivity of wild-type vector. More preferably, thecross-reactivity is essentially absent. Reducing cross-reactivity and/orNAb titers may be carried out by engineering AAV capsid proteins tocreate chimeric and/or altered rAAV vectors. Several methods are knownin the art to engineer genes, e.g., capsid genes, including, but notlimited to, DNA shuffling (family or single gene) (see Li et al.,Crameri, A et al. (1998). “DNA shuffling of a family of genes fromdiverse species accelerates directed evolution.” Nature 391:288-291, andStemmer, W P (1994). “Rapid evolution of a protein in vitro by DNAshuffling.” Nature 370: 389-391, all hereby incorporated by reference intheir entirety), site-directed mutagenesis, error-prone PCR (Moore, G.L., Maranas, C. D., 2000. “Modeling DNA mutation and recombination fordirected evolution experiments.” J. Theor. Biol. 205, 483-503,incorporated herein by reference), generating chimeras, or combinationsthereof such as a staggered extension process which is a method thatincorporates DNA shuffling and error-prone PCR techniques (Maheshri, Net al. (2006). “Directed evolution of adeno-associated virus yieldsenhanced gene delivery vectors.” Nat Biotechnol 24:198-204, hereinincorporated by reference in its entirety). Once the capsid proteinsundergo engineering, rAAV vectors are assayed for the desirableproperties for which one is selecting or screening. For example,selections and/or screens may be used to isolate rAAV clones that haveincorporated properties that produce a reduction in reactivity orcross-reactivity to NAbs. As described in Li et al., altered rAAVvectors were produced after using the DNA shuffling technique on thegenes responsible for encoding the AAV capsid proteins (i.e., the capgenes). The engineered rAAV clone that the authors created was producedfrom a combination of multiple serotypes and thus contained genomicfragments representing various parental serotypes. In order to assessthe immunological profile of the altered rAAV vector, it was subjectedto a series of crossreactivity tests. In these tests, antisera was takenfrom mice that were immunized with a particular AAV serotype (i.e., aparental serotype) from which the engineered rAAV vector was derived.The assays, in the Li et al. study, assessed the NAb titers and theextent to which the engineered rAAV vector cross-reacted with antiseragenerated from mice that were immunized with the AAV parental serotypes.Results showed that antisera from 3 of the 4 parental serotypes did notcross-react with the engineered rAAV clone while the remaining sampleshowed a 25-fold lower NAb titer.

In another embodiment, AAV vectors can be engineered for increasedtransduction efficiency and/or specificity. Increasing transductionefficiency and/or specificity may be carried out by engineering AAVcapsid proteins to create chimeric and/or altered rAAV vectors. Severalmethods are known in the art to engineer genes including, but notlimited to, DNA shuffling (family or single gene), site-directedmutagenesis, error-prone PCR, generating chimeras, or combinationshereof such as a staggered extension process which is a technique thatincorporates DNA shuffling and error-prone PCR. Once the capsid proteinsundergo engineering, rAAV vectors are assayed for the desirableproperties for which one is assaying. For example, selections and/orscreens may be used to isolate rAAV variants that have been endowed withan increase in efficiency and/or specificity for the targeted tissue(s)or cells.

Therapeutic Effect

In a preferred embodiment, the infusion of the therapeutic agentsdisclosed herein are used to achieve a therapeutic effect in a patientsuffering from cardiac disease. The treated individual may be monitoredfor clinical features which accompany the cardiac disorder to determineif a therapeutic effect is achieved. For example, subjects may bemonitored for reduction in adverse signs and symptoms associated withcardiovascular disease. For example, after treatment of congestive heartfailure in a subject using methods disclosed herein, the subject may beassessed for improvements in a number of parameters including, but notlimited to, increased lateral ventricle fractional shortening, augmentedcardiac contractility at the cellular and intact animal levels, reversalof cardiac remodeling, and normalization of the abnormally highdiastolic levels of cytosolic calcium. Other clinical and cardiacparameters which can be monitored in a subject treated with the presenttechnology include without limitation survival, cardiac metabolism,heart contractility, heart rate, ventricular function (e.g., leftventricular end-diastolic pressure (LVEDP), left ventricular systolicpressure (LVSP)), Ca²⁺ metabolism (e.g., intracellular Ca²⁺concentration, peak or resting [Ca²⁺], SR Ca²⁺ ATPase activity,phosphorylation state of phospholamban), force generation, relaxation, aforce-frequency relationship, cardiomocyte survival or apoptosis or ionchannel activity (e.g., sodium calcium exchange, sodium channelactivity, calcium channel activity, sodium potassium ATPase pumpactivity), activity of myosin heavy chain, BNP and NT-proBNP, troponinI, troponin C, troponin T, CK-MB, tropomyosin, actin, myosin light chainkinase, myosin light chain 1, myosin light chain 2 or myosin light chain3, IGF-1 receptor, PI3 kinase, AKT kinase, sodium-calcium exchanger,calcium channel (L and T), calsequestrin, or calreticulin. Theevaluation can be performed before, after, and during the treatment.Other measures of cardiac disease which can be monitored includefractional shortening, cardiac output, ejection fraction, Tau,regurgitant volume, number of hospital stays, quality of life, andtreadmill time, distance walked during 6 minute walk test, and maximaloxygen consumption (VO₂max). In one embodiment, patients may bemonitored with molecular biological techniques known in the art tomeasure the rAAV vector DNA, RNA, and/or proteins present in the cellsand/or tissues. In some embodiments, one can assess the copy number ofthe transgene per cell, the expression of the transgene at the mRNAand/or protein level per cell or in the tissue, and/or the percentage ofcells of a particular tissue, e.g. cardiomyocytes, being transfected.

In a preferred embodiment, the administration of a vasodilatingsubstance, preferably a NO increasing substance as described herein,more preferably nitroglycerin, increases the efficiency of transductionof the therapeutic agent. In some embodiments, the administration of avasodilator or NO increasing substance improves the therapeutic effectachieved by administration of the therapeutic agent alone, wherein thetherapeutic effect is monitored as described herein. In someembodiments, the administration of a vasodilator or NO increasingsubstance results in improved efficacy of the therapeutic agent, suchthat the same level of therapeutic effect can be achieved with lesstherapeutic agent. The improved efficacy can result in less therapeuticagent being needed in a single administration, or in feweradministrations of the therapeutic agent over time. In some embodiments,the administration of a vasodilating substance improves the therapeuticeffect achieved by administration of the therapeutic agent alone byincreasing the duration of the therapeutic effect. In some embodiments,the improvement in the transduction efficiency, therapeutic effect, orefficacy of the therapeutic agent is, is about, is at least, is at leastabout, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,90, 100, 125, 150, 175, or 200%, or 2, 3, 4, 5, 7, 10, 15, or 20 times,or a range defined by any two of the preceding values, when compared tothe value achieved without the administration of the vasodilatingsubstance. For example, if an increase in ejection fraction is observedto last for 3 months following treatment without the vasodilator, a 50%increase in the therapeutic effect following treatment with thevasodilator would result in an increase in ejection fraction that lastsfor 4.5 months.

The disclosed methods and therapeutic agents disclosed herein can becombined with existing treatments for cardiac disease, such as drugs orsurgical intervention, to provide an enhanced therapeutic effectcompared to existing treatments alone. An enhanced therapeutic effectmay be demonstrated by, for example, an extension of the time periodbetween the worsening of the signs or symptoms of the disease comparedto the average or typical time period for existing treatment regimens,or the lengthening of time required before additional treatment isrequired compared to the average or typical time for standard treatmentalone.

Kits

Other embodiments contemplated herein are kits comprising a container ofa therapeutic agent, for example a viral vector, and more preferably aAAV2/1/SERCA2a vector, and a container of a vasodilating substance orsubstances. In some embodiments, the kit contains a stock amount oftherapeutic agent and a carrier solution for dissolving or diluting thestock amount. In some embodiments, the kit contains a stock amount ofthe vasodilator(s), including but not limited to NO increasingsubstance(s), and a carrier solution for dissolving or diluting thestock amount. In some embodiments, the kit contains a container with amixture of the therapeutic agent and the amount of vasodilatingsubstance(s). The stock amounts of the therapeutic agent and/orvasodilating substance(s) can be in dry form requiring dissolution ormixing in a carrier solution, a concentrated solution requiringdilution, or in a form ready for administration to the patient withoutadditional preparation. In some embodiments, the kit includes one ormore intravascular infusion or injection catheters for intracoronaryadministration of the vasodilating substance(s) and/or therapeuticagent. In some embodiments, the kit includes one or more devices foradministration of the compounds in the kit through the catheter, forexample, a syringe. The kit can also include instructions foradministration of the viral vector and/or therapeutic agent, asdescribed in any of the embodiments disclosed herein, preferably byinfusion directly into the coronary circulation.

Embodiments of the invention will now be further described in thefollowing non-limiting examples. All references disclosed herein,including patents and non-patent literature, are hereby incorporated byreference in their entirety, and specifically for the disclosure thereofmentioned herein.

EXAMPLE 1 A 30 Day, Single Dose, Tissue Distribution Study of DirectIntracoronary Infusion of AAV1/SERCA2a (MYDICAR(®) in Normal GöttingenMinipigs

I. PROTOCOL SYNOPSIS Protocol Synopsis Title: A 30 Day, Single Dose,Tissue Distribution Study of Direct Intracoronary Infusion ofAAV1/SERCA2a (MYDICAR ®) in Normal Göttingen Minipigs Purpose: Todemonstrate the effect of pre-treatment with vasodilating agents such asnitroglycerin on AAV1/SERCA2a persistence in cardiac tissue at 30 daysfollowing a single administration of AAV1/SERCA2a (MYDICAR ®) inGöttingen minipigs with <1:2 baseline titer of anti- AAV1 neutralizingantibodies Study Design See Table 1 below Animal Model, Normal male andfemale Göttingen minipigs Species & Sex: Test Product: Vehicle control(CEL-1 formulation buffer) AAV1/SERCA2a test article Dosage: 10¹³ DRPAAV1/SERCA2a total dose (≈10¹² DRP/kg); Normal saline control Route ofAAV1/SERCA2a: Antegrade epicardial coronary artery infusionAdministration: Nitroglycerin Administration Group 3: 50 μgnitroglycerin was administered as a bolus intracoronary injectionimmediately prior to administration of AAV1/SERCA2a (see below “Methodof Administration”) Nitroglycerin Administration Group 4: 50 μgnitroglycerin was administered as a bolus intracoronary injectionimmediately prior to administration of AAV1/SERCA2a, and 100 μgnitroglycerin was also co-administered with AAV1/SERCA2a No otherpermeability enhancers are administered to any group (see below “Methodof Administration”) Total Sample 26 animals total (see Table 1) Size:Study Duration: 30 days total: dosing on Day 0, sacrifice at 30 dayspost-dosing Assessments: The following assessments were performed:During infusion: Vital signs and hemodynamic monitoring (blood pressure,heart rate and rhythm, respiratory rate, and O2 pulse oximeter, andend-tidal carbon dioxide). Hematology/coagulation parameters Clinicalchemistry parameters Cardiac enzymes (creatine kinase (CK) (total andisoenzymes BB, MB, and MM) and troponin I) AAV1 neutralizing antibodypre-screening Expression of SERCA2a in cardiac tissue via RT-PCR andWestern blot Body and organ weights Gross or macroscopic findingsExpression of SERCA2a mRNA expression was assessed via RT-PCR in theheart SERCA2a: (12 regions) at terminal sacrifice on Day 30 SERCA2aprotein expression was assessed via western blot in the heart (12regions) at terminal sacrifice on Day 30 in three animals from Group 1,three animals from Group 3, and one animal from Group 5.

TABLE 1 Group Designation and Dose Levels Nitroglycerin NitroglycerinInfusion Pre-Treat Co-Admin Duration Test Saline Treatment Group Total(μg) (μg) (min) Article Control Group #1 No nitroglycerin pre-treatment/6 No No 10 ✓ AAV1/SERCA2a 10 min infusion Group #2 No nitroglycerinpre-treatment/ 6 No No 4 ✓ AAV1/SERCA2a 4 min infusion Group #3Pre-treat 50 μg nitroglycerin/ 6 50 No 10 ✓ AAV1/SERCA2a 10 min infusionGroup #4 Pre-treat 50 μg nitroglycerin/ 6 50 100 10 ✓ AAV1/SERCA2a + 100μg nitroglycerin 10 min infusion Group #5 No nitroglycerinpre-treatment/ 2 No No 10 ✓ Saline Control 10 min infusion

Method of Administration

Administration on Day 0 was via the direct coronary infusion proceduredescribed below. Animals from Groups 1-3 were dosed with a total volumeof 12 mL of AAV1/SERCA2a solution which was infused at a constant rateof 1.2 mL/minute over a 10 minute period. Animals from Groups 3 and 4received nitroglycerin as a bolus intracoronary injection immediatelyprior to administration of AAV1/SERCA2a. Animals from Group 3 were dosedwith a total volume of 12 mL of AAV1/SERCA2a and nitroglycerin solution,which was infused at a constant rate of 1.2 mL/minute over a 10 minuteperiod. Animals from Group 4 were dosed with a total volume of 12 mL ofnormal saline, which was infused at a constant rate of 1.2 mL/minuteover a 10 minute period. The direct infusion system is composed ofstandard (commercially available) components including a conventionalguide sheath, 0.014″ guide-wire, a 5F infusion (guide) catheter and twoprogrammable syringe pumps. The direct intracoronary infusion procedurebegan with introduction of the conventional guide sheath using a commoncarotid arterial or femoral arterial approach. The Coronary InfusionCatheter (e.g., Cordis Vista Brite Tip Guiding Catheter or similar modelappropriate for cannulation of the left main coronary artery) was thenplaced in the left main coronary artery under fluoroscopic guidance.Once the catheter was in place, it was connected to the firstprogrammable syringe pump (e.g., NE-1000 Programmable Syringe Pump, NewEra Pump Systems) using standard tubing and purging technique. TheAAV1/SERCA2a was then delivered, followed by a sterile saline flush ofthe catheter dead volume with the second programmable syringe pump.

Results

Results show substantial improvement in one or more biologicalactivity/efficacy measures in the subjects that were treated withAAV1/SERCA2a and nitroglycerin compared to the subjects that weretreated with AAV1/SERCA2a without nitroglycerin. SERCA2a expression wasassessed via RT-PCR (mRNA) and Western blot (protein) in the heart (LVfree wall, LV posterior (inferior) segment, and LV anterior segment) atterminal sacrifice on Day 30 from heart sections as shown in FIG. 1 (Mapof Heart Sections for Expression of SERCA2a: A (basal layer of LV): #1septum; #2 LV anterior wall; #3 LV free wall; #4 LV posterior wall; B(middle layer of LV): #5 septum; #6 LV anterior wall; #7 LV free wall;#8 LV posterior wall; C (apical layer of LV): #9 LV anterior wall; #10LV posterior wall; (RV): #11 RV free wall basal layer; #12 RV free wallapical layer). Total mRNA and protein expression in the left ventricleof individual sections was calculated (sum of sections 1-10 in FIG. 1),and the septum (section #5 in FIG. 1), and the middle layer anteriorwall (section #6 in FIG. 1) were also analyzed separately, as theseregions represent deep myocardium, furthest from the site of infusion.Obtaining adequate gene transfer into these deep myocardial regions ismost difficult; however, administration of nitroglycerin enhanced vectoruptake, especially in these territories. The results below showimprovement in expression of SERCA2a mRNA and protein when AAV1/SERCA2awas administered in combination with nitroglycerin, especially in the LVMid-Anterior wall and Septum, see FIGS. 2, and 3, respectively. Theincrease of mRNA and protein shown in these figures are in addition tothe normal background levels of SERCA2a mRNA and protein. This is incontrast to humans suffering from cardiac failure, wherein SERCA2aexpression levels are below normal levels. Thus, it is expected that theaffect of AAV1/SERCA2a treatment on SERCA2a expression levels inpatients with cardiac failure will be even more pronounced than theeffect reported in FIGS. 2 and 3, given the lower baseline level ofSERCA2a expression.

Safety

The mean aortic pressures from Groups 3 and 4 are shown in FIG. 4. Thechanges seen are for the most part within what would be expected overthe course of an experiment. Note that there was a maximum of 6 mm Hgdecrease in Mean arterial pressure (MAP) compared to baseline and exceptfor one time point in group 4, all other changes for nitroglycerintreated animals were within that same range. A modest decrease in MAPsuch as this is not meaningful and poses no safety concern. Bycomparison, Sasano et al. reported a decrease in blood pressure of 30mmHg after infusion of the pretreatment and virus solutions. Moreover,subjects in the Sasano study experienced a decrease in heart rate thataveraged about 50-60/minute. Authors report that the blood pressure andheart rate stabilized within the first minute of perfusion.Nevertheless, ventricular fibrillation (VF) occurred during coronaryinfusion at a 50% rate for the first 10 pigs but dropped to 5.6% for theremaining 71 pigs.

EXAMPLE 2 Preliminary Phase I Clinical Results—Effect of Nitroglycerinon Endpoint Measure of Cardiac Function in Patients receiving MYDICAR®

Protocol Synopsis Protocol CELL-001 Number: Title: A Phase 1 Trial ofIntracoronary Administration of MYDICAR ® (AAV1/SERCA2a) in Subjectswith Heart Failure Divided into Two Stages: Stage One —Open-Label,Sequential Dose-Escalation Cohorts Followed in Stage Two —by Randomized,Double-Blind, Placebo-Control, Parallel Cohorts Short Title: CalciumUp-Regulation by Percutaneous Administration of Gene therapy In CardiacDisease (CUPID Trial) Development 1 Phase: Objectives: To demonstratethe safety and feasibility of a single antegrade epicardial coronaryartery infusion of 4 dose levels of AAV1 vector expressing the transgenefor SERCA2a to subjects with ischemic or non-ischemic cardiomyopathy andNYHA Class III/IV symptoms of heart failure To demonstrate theactivity/efficacy of MYDICAR ® in order to validate appropriate doselevels for future studies Study This is a phase 1, multi-center,two-stage trial of a single intracoronary Design: administration ofAAV1/SERCA2a (MYDICAR ®). In Stage 1, 12 subjects, 3 subjects each in 4sequential dose escalation cohorts, will be enrolled and treated withopen-label MYDICAR ®. The rate of enrolling and treating subjects willbe controlled as follows: Within Cohorts 1 and 2, a subject will beobserved for a minimum of 1 week before treating the next subject inthat cohort. For Cohorts 3 and 4, the first subject will be observed fora minimum of 6 weeks before treating the second subject, and the secondsubject will be observed for a minimum of 1 week before treating thethird subject. In order to advance to the next cohort, 2 of the 3subjects will be observed for a minimum of 90 days and the third subjectfor a minimum of 30 days and their safety data reviewed by the DataMonitoring Committee (DMC). After all subjects in Stage 1 are enrolledand 2 of the 3 subjects in the fourth cohort have been observed for aminimum of 90 days and the third subject for a minimum of 30 days, theDMC will review all safety data available at that time and makerecommendations for opening Stage 2 of the trial. An additional 33 or 34subjects may be enrolled in Stage 2 of the trial in a double-blinded,parallel cohort design. Subjects will be treated with one of 2 or 3doses of MYDICAR ® or placebo. The dose groups will be recommended bythe DMC based on an assessment of all safety and efficacy data availablefrom Stage 1 subjects at that time. If 3 doses are recommended, subjectswill be randomized in a ratio of 8:8:8:9(MYDICAR ®:MYDICAR ®:MYDICAR ®:placebo). If 2 doses are recommended,subjects will be randomized in a ratio of 11:11:12(MYDICAR ®:MYDICAR ®:placebo). All subjects in Stages 1 and 2 will beseen at screening over 2 visits, Day 0 for investigational productadministration followed by continuous observation in the hospital for18-24 hours post-dose; at weeks 1, 2, 3, 4, 5 and 6; and at months 2, 3,6, 9 and 12. All subjects will then undergo long-term follow-up viasemi-structured telephone questionnaires every 6 months for anadditional 2 years. Number of A total of 34 to 36 subjects will betreated with MYDICAR ® with or without Subjects: nitroglycerin, and 9 to12 subjects will be treated with placebo with or without nitroglycerin.In Stage 1, 12 subjects will be treated with open-label MYDICAR ® withor without nitroglycerin. In Stage 2, 24 subjects will be randomized toMYDICAR ® with or without nitroglycerin and 9 subjects will berandomized to placebo with or without nitroglycerin if 3 doses ofMYDICAR ® are evaluated, or 22 subjects will be randomized to MYDICAR ®with or without nitroglycerin and 12 subjects will be randomized toplacebo with or without nitroglycerin if 2 doses of MYDICAR ® arestudied. Method of In Stage 1, all subjects will be assigned open-labelMYDICAR ® with or Subject without nitroglycerin in all 4 cohorts. InStage 2, subjects will be randomized Assignment: centrally in a ratio of8:8:8:9 (MYDICAR ®:MYDICAR ®:MYDICAR ®:placebo, all with or withoutnitroglycerin) if 3 doses MYDICAR ™ are selected or in a ratio of11:11:12 (MYDICAR ®:MYDICAR ®:placebo, all with or withoutnitroglycerin) if 2 doses of MYDICAR ® are selected. No. of Study Up to9 centers in the United States Centers: Diagnosis: Adult subjects withNYHA Class III/IV chronic heart failure due to ischemic or non-ischemiccardiomyopathy Inclusion Screening should be performed within 30 daysprior to administration of Criteria: MYDICAR ® or placebo on Day 0. 1.Age 18-75 years of age 2. Chronic ischemic or non-ischemiccardiomyopathy. Subjects with ischemic cardiomyopathy should have atleast one major coronary vessel with TIMI grade 3 flow. 3. Leftventricular ejection fraction (LVEF) ≦30% 4. Diagnosis of NYHA ClassIII/IV heart failure for a minimum of 6 months prior to enrollment 5.Maximal oxygen consumption (VO₂ max) ≦16 mL/kg/min within 90 days priorto enrollment 6. An implantable cardioverter defibrillator (ICD)implanted a minimum of 30 days prior to enrollment 7. Treatment withappropriate heart failure therapy as tolerated, including, but notlimited to: a. Medical therapy as tolerated, including angiotensinreceptor blocker and/or angiotensin receptor converting enzymeinhibitor, beta blocker and aldosterone antagonist. Dosing of the abovemedications should be stable for a minimum of 30 days prior toenrollment; and/or b. Resynchronization therapy, if clinicallyindicated, should have been implanted at least 6 months prior toenrollment 8. All women of childbearing potential should have a negativeurine pregnancy test prior to administration of investigational productand agree to use adequate contraception (defined as oral or injectablecontraceptives, intrauterine devices, surgical sterilization or acombination of a condom and spermicide) or limit sexual activity tovasectomized partner for 3 months after administration ofinvestigational product. Men capable of fathering a child should agreeto use barrier contraception (combination of a condom and spermicide) orlimit activity to post-menopausal, surgically sterilized, or acontraception-practicing partner, for 3 months after administration ofinvestigational product. 9. Ability to sign Informed Consent Form (ICF)and Release of Medical Information Form Exclusion 1. Any intravenoustherapy with positive inotropes, vasodilators, or diuretics Criteria:within 30 days prior to enrollment 2. Restrictive cardiomyopathy,obstructive cardiomyopathy, pericardial disease, amyloidosis,infiltrative cardiomyopathy, uncorrected thyroid disease, or dyskineticLV aneurysm 3. Cardiac surgery, percutaneous coronary intervention, orvalvuloplasty within 30 days prior to enrollment 4. Clinicallysignificant myocardial infarction (e.g., ST elevation MI [STEMI] orlarge non-STEMI) within 6 months prior to enrollment 5. Prior hearttransplantation, left ventricular reduction surgery (LVRS),cardiomyoplasty, passive restraint device (e.g., CorCap ™ CardiacSupport Device), surgically implanted LVAD or cardiac shunt 6. Likely toreceive cardiac resynchronization therapy, cardiomyoplasty, LVRS, hearttransplant, conventional revascularization procedure, or valvular repairwithin 6 months following enrollment 7. Prior coronary artery bypassgraft(s) (CABG) 8. Exercise capacity primarily limited by obesity,peripheral vascular disease, or orthopedic problems and not byunderlying heart failure 9. Known hypersensitivity to octafluoropropane(component of the intravenous echocardiography contrast agent,DEFINITY ®) or other contrast dyes used for angiography; history of, orlikely need for, high dose steroid pretreatment prior to contrastangiography 10. Significant left main or ostial right coronary lumenalstenosis in the opinion of the investigator 11. Expected survival <1year in the investigator's medical opinion 12. Suspected or activeviral, bacterial, fungal, or parasitic infection within 48 hours priorto enrollment 13. Liver function tests (ALT, AST, alkalinephosphatase) >2x Upper Limit of Normal (ULN) within 30 days prior toenrollment or known intrinsic liver disease (e.g., cirrhosis, chronichepatitis B or hepatitis C virus infection) 14. Current or likely needfor hemodialysis within 12 months following enrollment 15. Bleedingdiathesis or thrombocytopenia defined as platelet count <50,000platelets/μL 16. Anemia defined as hemoglobin <10 g/dL 17. Known AIDS orHIV-positive status, or a previous diagnosis of immunodeficiency with anabsolute neutrophil count <1000 cells/mm³ 18. Previous participation ina study of gene transfer 19. Presence of neutralizing anti-AAV1antibodies at titer ≧1:4 within 6 months of screening 20. Receivinginvestigational intervention or participating in another clinical studywithin 30 days or within 5 half-lives of the investigational drugadministration prior to enrollment 21. Pregnancy or lactation 22. Recenthistory of psychiatric disease (including drug or alcohol abuse) that islikely to impair subject's ability to comply with protocol-mandatedprocedures, in the opinion of the investigator Test MYDICAR ®(AAV1/SERCA2a) or matching placebo, all with or without Product:nitroglycerin. Dose: Single Administration of MYDICAR ®: 1.4 × 10¹¹DNase Resistant Particles (DRP) AAV1/SERCA2a (~2 × 10⁹ DRP/kg*) 6 × 10¹¹DRP AAV1/SERCA2a (~8.6 × 10⁹ DRP/kg*) 3 × 10¹² DRP AAV1/SERCA2a (~4.3 ×10¹⁰ DRP/kg*) 1 × 10¹³ DRP AAV1/SERCA2a (~1.4 × 10¹¹ DRP/kg*) Matchingplacebo * Assuming a 70 kg individual Some subjects to receive 150 μg(total dose) of nitroglycerin. Mode of MYDICAR ®: Antegrade epicardialcoronary artery infusion into left coronary Administration: arteryand/or right coronary artery via percutaneous catheter over 10 minutes.Nitroglycerin: Antegrade epicardial coronary artery infusion into leftcoronary artery and/or right coronary artery via percutaneous catheterover a period of less than one minute using 1.5 cc of a 100 μg/mLsolution of nitroglycerin, just prior to administration of MYDICAR ®.Alternatively, nitroglycerin was given PO + IV where PO administrationwas given at 0.4 mg before MYDICAR ® administration and by IV at 20μg/minute up to 691.3 μg before and during MYDICAR ® administration.Nitroglycerin was also given in an IV regimen at 20 μg/minute up to 2118μg before and during MYDICAR ® administration. IV nitroglycerin wasterminated after administration of MYDICAR ®. No othervasodilators/permeability enhancers are administered. Duration ofTreatment consists of a single dose administration of MYDICAR ® with orTreatment: without nitroglycerin or placebo with or withoutnitroglycerin followed by 12 months of observation. After completion ofthe 12 months, subjects will receive a follow-up telephone call every 6months for an additional 2 years to elicit information abouthospitalizations, new medical conditions, heart failure status, and longterm survival. Safety Physical examination Assessments: Follow-uphistory: interim illnesses, number of days hospitalized, and adverseevents Complete blood count: WBC and differential, hemoglobin,hematocrit, platelet count Blood chemistries: electrolytes, blood ureanitrogen, creatinine, total bilirubin, alkaline phosphatase, alanineaminotransferase, aspartate aminotransferase, albumin, and lactatedehydrogenase Urinalysis Creatine kinase-MB, troponin T ELISPOT (anexperimental immunologic function assay) Echocardiogram Interrogation ofICD ECG Primary Echocardiographic assessments (including contrastechocardiography) Activity/ including LVEF, LV dimensions, regional wallmotion, diastolic function, and Efficacy mitral regurgitationAssessments: VO₂ max measured during cardiopulmonary exercise testing6-minute walk test N-terminal prohormone brain natriuretic peptide(NT-proBNP) NYHA classification Minnesota Living with Heart FailureQuestionnaire (MLWHFQ) Safety DMC Oversight: Morbidity & MortalityCommittee Primary The primary endpoint is safety as measured by theincidence and severity of Endpoint: adverse events, including all-causemortality, progression of heart failure (HF) leading to hospitalization,and/or IV inotrope, vasodilator, and/or diuretics administration. Thepercentage of subjects experiencing an event will be calculated forsurvivors and for all subjects enrolled. Frequency of all-causehospitalizations and cardiovascular hospitalizations during the 12months following intervention will also be analyzed. The total length ofstay (hospital days) will be tabulated. All deaths and hospitalizationswill be classified by the blinded independent Morbidity & MortalityCommittee, distinguishing between the primary cause and immediateunderlying cause of death or hospitalization. Details including the dateof death, immediate cause of death, underlying cause of death, notationof autopsy being performed, and clinical narrative of the event will bereported to the Sponsor as soon as feasible. The primaryactivity/efficacy endpoints to be evaluated and compared within andbetween treatment groups based on changes from baseline to 3, 6, 9 and12 months following investigational product administration include thefollowing: VO₂ max assessed by cardiopulmonary exercise testing Distancewalked during the 6-minute walk test Echocardiographic assessmentsincluding left ventricular ejection fraction, LV dimensions, regionalwall motion, diastolic function, and mitral regurgitation NT-proBNPlevel NYHA Classification Quality of Life assessed by Minnesota Livingwith Heart Failure Questionnaire (MLWHFQ) In addition, echo measurementswill be analyzed at one month. Safety analyses will be performed for allsubjects enrolled in the study and administered the test article.Primary efficacy analysis will be performed for stage 2 subjects.Additional analyses will be performed for stage 1 and 2 subjectscombined according to the respective dose levels. Statistical The twoprincipal aims of the study, in addition to demonstrating feasibility,are Methods: to demonstrate the safety of MYDICAR ® administration,validate the preliminary estimate of clinical activity, and verifyappropriate dose levels for future studies of MYDICAR ®. Safety will bedemonstrated based on the incidence of adverse events. Stage 1 studydata will be analyzed descriptively. Primary efficacy analysis will beperformed using Stage 2 data at 6 months after test articleadministration. Secondary analysis based on the 3- and 12-month data andadditional analysis based on the combined data for Stage 1 and Stage 2subjects who were administered the same dose will be performed. Activitywill be measured by a set of pre-specified endpoints including VO₂ max,LV function measured by echocardiography, 6-minute walk test, NYHAclassification, NT-proBNP level and MLWHFQ. Treatment success will beevaluated based on the trends in between-group comparisons for 5efficacy domains: 1. LV function and remodeling (Ejection Fraction, EndSystolic Volume, other echocardiography parameters) 2. Symptomatic(NYHA, MLWHFQ) 3. Functional (6-minute walk test, VO₂ max) 4. Biomarkers(NT-proBNP) 5. Clinical outcome (all-cause deaths, HF hospitalizationsor emergency room visits, IV inotropes/vasodilators/diuretics due to HFprogression) Numerical and graphical summary measures (including mean,median, standard deviation, range, histograms, and scatterplots, etc.)will be used to describe these endpoints in each treatment group atbaseline, and at 1, 3, 6, 9 and 12 months post-enrollment. Mixed effectsmodels will be used to estimate changes in each endpoint over timewithin each treatment group, and to estimate the difference in changesover time between treatment groups. Percent change from baseline to eachtime point in continuous variables will be analyzed descriptively. Bothpoint estimates and 95% confidence intervals will be computed.Proportions of patients categorized as “improved”, “no change” and“worsened” based on activity parameters observed at 1, 3, 6, and 12months will be compared across treatment groups.

Results

Table 2 below shows the preliminary results of this study. These resultsdemonstrate a substantial and significant improvement in one or moreactivity/efficacy endpoints in the subjects that were treated withMYDICAR® and nitroglycerin compared to the subjects that were treatedwith MYDICAR® without nitroglycerin. For other details, see Hajjar etal., Journal of Cardiac Failure, 2008 14(5); 355-67, incorporated hereinby reference in its entirety.

TABLE 2 Nitroglycerin Prior To Absolute Ejection Fraction Infusion (EF)(%) LCA, RCA, EF Change EF Change/ IC, μg IC, μg Other Baseline 2 Mo 6Mo at 6 mo Patient Status Subject 1 — — — 18 19 16 −2 Worsened/ w/o NO(Heart Transplant >6 mo) Subject 4 — — — 16 13 — −3 Worsened/ w/o NO(Died >3 mo) Subject 3 — 150 — 30 29 33 +3 Improved/ with NO ImprovedSubject 2 — — 20 μg/min 25 32 31 +6 Improved/ with NO IV, 2118 μgImproved total Subject 5 — — 20 μg/min 20 20 21 +1 Slight with NO IV,691 μg Improvement/ total + 0.4 mg Improved PO Subject 6 — — — 25 24 26+1 Slight w/o NO Improvement/ Improved

What is claimed is:
 1. A method of treating or preventing acardiovascular disease by transfecting cardiac cells of a large mammal,the method comprising: identifying a mammal in need of treatment orprevention of a cardiovascular disease; administering a vasodilatingsubstance to said mammal sufficient to dilate a blood vessel of thecoronary circulation; and administering a therapeutic polynucleotideinto a blood vessel of the coronary circulation in vivo; wherein saidtherapeutic polynucleotide is infused into said blood vessel over aperiod of at least about three minutes, wherein the coronary circulationis not isolated or substantially isolated from the systemic circulationof the mammal, and wherein said therapeutic polynucleotide transfectscardiac cells of said mammal resulting in the treatment or prevention ofsaid cardiovascular disease.
 2. The method of claim 1, wherein saidvasodilating substance is a nitric oxide (NO) increasing substance. 3.The method of claim 2, wherein said NO increasing substance isnitroglycerin.
 4. The method of claim 2, wherein said NO increasingsubstance is administered into a blood vessel of the coronarycirculation.
 5. The method of claim 4, wherein said NO increasingsubstance is administered in a manner selected from the group consistingof: prior to said infusion of said therapeutic polynucleotide,concurrently with said infusion of said therapeutic polynucleotide, andprior to and concurrently with said infusion of said therapeuticpolynucleotide.
 6. The method of claim 4, wherein said NO increasingsubstance is administered as a bolus injection not more than 5 minutesprior to said infusion of said therapeutic polynucleotide.
 7. The methodof claim 4, wherein said NO increasing substance is administered as abolus injection not more than 5 minutes prior to said infusion of saidtherapeutic polynucleotide and wherein said NO increasing substance isinfused into said blood vessel concurrently with said infusion of saidtherapeutic polynucleotide over a period of at least about 10 minutes.8. The method of claim 4, wherein the NO increasing substance is about50 μg to about 150 μg of nitroglycerin.
 9. The method of claim 4,wherein said administration of said NO increasing substance comprisesantegrade epicardial coronary artery injection of 1.5 mL of a 100 μg/mLsolution of nitroglycerin into at least one of a left or right coronaryartery via percutaneous catheter over a period of less than 1 minute,wherein said administration of said NO increasing substance is less than3 minutes prior to said infusion of said therapeutic polynucleotide, andwherein no other vasodilator or vascular permeation enhancer isadministered to said mammal.
 10. The method of claim 9, furthercomprising infusing nitroglycerin into said blood vessel concurrentlywith said infusion of said therapeutic polynucleotide.
 11. The method ofclaim 9, wherein said mammal is a human and said cardiovascular diseaseis heart failure, wherein said therapeutic polynucleotide is packaged ina DNAse resistant particle (DRP) of a AAV2/1 viral vector, and a totalnumber of DRP infused into said blood vessel is not more than about1×10¹³, wherein the therapeutic polynucleotide comprises a SERCA2acoding sequence, wherein said blood vessel is at least one of the leftor right coronary artery, and wherein said infusion of said therapeuticpolynucleotide lasts at least about 10 minutes.
 12. The method of claim11, wherein said treatment improves a measurement of absolute ejectionfraction of said human's heart six months after said treatment ascompared to a measurement of absolute ejection fraction of said human'sheart prior to said treatment.
 13. The method of claim 2, wherein saidNO increasing substance is administered systemically.
 14. The method ofclaim 13, wherein said NO increasing substance is administeredsystemically in a manner selected from the group consisting of:intravenous injection, intravenous infusion, oral administration,transdermal administration, and subcutaneous administration.
 15. Themethod of claim 14, wherein said NO increasing substance is administeredin a manner selected from the group consisting of: prior to saidinfusion of said therapeutic polynucleotide, concurrently with saidinfusion of said therapeutic polynucleotide, and prior to andconcurrently with said infusion of said therapeutic polynucleotide. 16.The method of claim 13, wherein said NO increasing substance isnitroglycerin.
 17. The method of claim 16, wherein about 0.5 mg to about2.5 mg of nitroglycerin is administered by intravenous infusion over aperiod of at least 30 minutes prior to said infusion of said therapeuticpolynucleotide, wherein said infusion of said therapeutic polynucleotidebegins within not more than three minutes of the completion of saidintravenous infusion of nitroglycerin, and wherein no other vasodilatoror vascular permeation enhancer is administered to said mammal.
 18. Themethod of claim 17, further comprising infusing an additional amount ofnitroglycerin concurrently with said infusion of said therapeuticpolynucleotide.
 19. The method of claim 17, wherein said mammal is ahuman and said cardiovascular disease is heart failure, wherein saidtherapeutic polynucleotide is packaged in a DNAse resistant particle(DRP) of a AAV2/1 viral vector, and a total number of DRP infused intosaid blood vessel is not more than about 1×10¹³, wherein the therapeuticpolynucleotide comprises a SERCA2a coding sequence, wherein said bloodvessel is at least one of the left or right coronary artery, and whereinsaid infusion of said therapeutic polynucleotide lasts at least about 10minutes.
 20. The method of claim 19, wherein said treatment improves ameasurement of absolute ejection fraction of said human's heart sixmonths after said treatment as compared to a measurement of absoluteejection fraction of said human's heart prior to said treatment.